Abs#1. The influence of Muc5b overexpression and aging on the development of lung fibrosis in mice
Kristen A. Ball1,2,*, Corinne Hennessey2,*, Evgenia Dobrinskikh2,3, David Schwartz1,2,**
1. University of Colorado Anschutz Medical Campus, Skaggs School of Pharmacy and Pharmaceutical Sciences
2. University of Colorado Anschutz Medical Campus, Division of Pulmonary Sciences and Critical Care Medicine
3. University of Colorado Anschutz Medical Campus, Department of Pediatrics. *: Shared primary author. **: Corresponding author
The dominant risk factor for the development of idiopathic pulmonary fibrosis (IPF) is the gain-of-function MUC5B promoter variant rs35705950. Heterozygous individuals (GT) have a 5-10 fold risk (OR) and homozygous individuals (TT) have a 10-20 fold risk (OR) for IPF. However, the pathogenesis of MUC5B-induced lung fibrosis has yet to be defined. The objective of this study was to identify if overexpression of Muc5b in mice induces fibrosis as mice reach old age (another key risk factor for IPF). Muc5b transgenic mice and their littermates were sacrificed at two different ages, 8-12 weeks and 18 months of age, and evaluated for their presence of lung fibrosis. Scgb1a1-Muc5bTg (club cell overexpression), SFTPC-Muc5bTg (alveolar epithelial type 2 cell overexpression), and Scgb1a1/SFTPC-Muc5bTg (overexpression of Muc5b in both club cells and alveolar epithelial type 2 cells) were compared with their wild type littermates for all conditions. Results demonstrate that Scgb1a1/SFTPC-Muc5bTg mice at 18 months of age had significantly more lung fibrosis than the other strains as demonstrated by second harmonic generation. There is also evidence that aging results in increased lung inflammation in all strains of mice, having the Scgb1a1/SFTPC-Muc5bTg mice demonstrating the most significant inflammation. These findings further support the concept that overexpression of Muc5b alone can cause lung fibrosis, and this is exacerbated with aging. Future research will investigate: 1) the impact of Muc5b expression and aging on gene expression and gene regulation, and 2) whether chronic inflammation and injury alters lung epithelial memory resulting in persistent tissue damage and repair.
Abs#2. Thinking Zinc: A nutritional intervention for metal toxicity
Erica Dashner-Titus1, Debra MacKenzie1, Chris Shuey2, Laurie G. Hudson1
1. Department of Pharmaceutical Sciences, Albuquerque, NM
2. Southwest Research and Information Center, Albuquerque, NM
Navajo communities have concerns about health consequences of chronic exposures to uranium, arsenic and other metals associated with living in proximity to abandoned uranium mines on Navajo Nation. We and others have demonstrated that certain metals, including arsenic and uranium bind with zinc finger proteins and disrupt normal functions leading to inhibition of DNA repair as well as immune dysregulation. Taking supplemental zinc has been shown in experimental models to reverse the effects of arsenic or uranium. Thinking Zinc is a one-armed intervention trial to test the hypothesis that zinc supplementation, at (but not exceeding) recommended adult dietary levels, can alleviate adverse effects of uranium and mixed metal exposures in populations living in contaminated areas on Navajo Nation. Baseline samples were provided at two time points before taking zinc and two time points after taking zinc. Longitudinal biomonitoring revealed patterns of episodic high metal exposures to uranium, vanadium and other metals in many study participants. More than 10% of the samples measured were higher than the 95th percentile of NHANES for antimony, platinum, strontium, tin and uranium. We saw a decrease in biomarkers of both inflammation and oxidative stress in study participants after taking supplemental zinc. Supplemental zinc decreased some measures of inflammation and oxidative stress, processes known to contribute to chronic diseases including autoimmunity and cancer. Fluctuations in exposure could provide important clues regarding exposure sources as well as inform models for toxicity testing.
Abs#3. The Effects of Ozone Exposure on Vasculature Inflammation is Dependent on Fatty Acid Diets
Brenna Baird
Department of Pharmaceutical Sciences, University of New Mexico
Ozone is a highly reactive gas with a structure that facilitates degradation of organic molecules. Ground-level ozone is created synthetically, through man-made pollution. Exposure to ground level ozone can induce a number of symptoms, such as irritated airways and decreased lung function. However, the mechanisms underlying the varying levels of symptoms is yet to be uncovered. Inhalation of ozone can result in the breakdown of organic molecules within the body causing a state of inflammation both locally and systemically. This state of inflammation may be facilitated by the type of diet most commonly consumed by an individual. Saturated and unsaturated fats differ in structure, which alters their susceptibility to interact with ozone. Saturated fats, such as coconut oil, have a structure that allows ozone to oxidize these molecules into free radicals, making them more reactive. This is the mechanism thought to lead to the inflammatory state after ozone exposure. A diet high in saturated fats may increase the likelihood of inflammatory patterns following ozone exposure. Brain inflammation after ozone exposure is of particular interest due to the impact on neurological function. We demonstrate the systemic impact of ozone exposure on brain inflammation is based on the type of diet consumed.
Abs#4. Cross-sectional and Longitudinal Serum PFAS Concentrations in Municipal Firefighters
Amy Nematollahi1, amilajo@email.arizona.edu; Julia M. Fisher2, julia@statlab.bio5.org; Melissa A. Furlong1, mfurlong@arizona.edu; Paloma I. Beamer1, pbeamer@arizona.edu; Jaclyn M. Goodrich3, gaydojac@umich.edu; Judith M. Graber4, judith.graber@rutgers.edu; Antonia M. Calafat5, aic7@cdc.gov; Julianne Cook Botelho5, gur5@cdc.gov; Shawn C. Beitel1, sbeitel@arizona.edu; Sally R. Littau1, srl@arizona.edu; John J. Gulotta6, john.gulotta@tucsonaz.gov; Darin D. Wallentine6, darin.wallentine@tucsonaz.gov; Jefferey L. Burgess1, jburgess@arizona.edu
1. Environmental Health Sciences, Mel and Enid Zuckerman School of Public Health, The University of Arizona, Tucson, Arizona, USA
2. BIO5 Institute, Statistics Consulting Laboratory, The University of Arizona, Tucson, Arizona, USA
3. University of Michigan School of Public Health, Ann Arbor, Michigan, USA
4. Rutgers School of Public Health, Rutgers University, Piscataway, New Jersey, USA
5. Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
6. Tucson Fire Department, Tucson, Arizona, USA
Background: Firefighters may be at occupational risk for increased serum per- and polyfluoroalkyl substances (PFAS) concentrations through exposure to PFAS in fire suppression foams, firefighting gear, and combustion products.
Objective: To compare serum PFAS concentrations in municipal incumbent and recruit firefighters and evaluate temporal trends among recruits.
Methods: Serum concentrations of nine legacy PFAS were measured in 101 incumbent and 55 recruit firefighters at enrollment, and 20-37 months later in the recruits. Cross-sectional measures of PFAS from incumbent and recruit firefighters were examined using linear and logistic regression models. Longitudinal associations of PFAS with cumulative fire exposures and most recent fire exposures in recruit firefighters were assessed using linear mixed effect and logistic regression models.
Results: Concentrations of linear perfluorooctanoate (n-PFOA) and perfluorononanoate (PFNA) were significantly lower in incumbents than recruits at enrollment. Temporal trends for recruits showed significant declines in linear perfluorooctane sulfonate (n-PFOS), monomethyl branched isomers of PFOS, n-PFOA, PFNA, and perfluorodecanoate (PFDA). Detection frequency increased for branched PFOA isomers and decreased for 2-(N-methyl-perfluorooctane sulfonamide) acetate (MeFOSAA). No significant associations with cumulative fire exposures were observed.
Significance: Legacy serum PFAS concentrations were not significantly higher in incumbent firefighters than recruits and were not associated with cumulative fireground exposure in recruits.
Abs#5. Dissecting the role of ?-catenin in primary hepatic stellate cell activation
Lauren Rutt, Rebecca McCullough
University of Colorado Anschutz Medical Campus
Chronic Liver Disease (CLD) accounts for nearly 2 million deaths annually and is described as non-resolving inflammation and fibrosis. Hepatic stellate cells (HSC) play a key role in the progression of liver fibrosis by secreting fibrogenic factors and the accumulation of extracellular matrix. Despite what is known about HSCs, detailed mechanisms involved in the activation and reversion of HSCs in liver fibrosis are not well understood. Wnt signaling is a signaling cascade that elicits diverse cellular responses via canonical ?-catenin-dependent and noncanonical, ?-catenin-independent signals and have been recently implicated in HSC activation during CLD. It is our working hypothesis that activators of ?-catenin promote hepatic stellate cell activation during the early pathogenesis of fibrosis in CLD. Primary mouse HSCs (mHSCs) were isolated from livers and enzymatically digested; mHSCs were collected using a Histodenz gradient and plated on plastic culture plates for ten days. mHSCs were challenged with or without exogenous of 10ng/ml of: TGF-?, Wnt3a, and Wnt5a for 48 hours. Mice were fed a pair-diet or ethanol-containing diet for 5 weeks in combination with administered carbon tetrachloride (CCl4) or olive oil as vehicle twice weekly at a dose of 1ul/g body weight; tissues were harvested 72hrs after the last CCl4 injection. mHSCs underwent culture-induced spontaneous activation over 10 days and were validated by immunofluorescent expression of Desmin and mRNA expression of col1a1 and ?-sma; expression of ?-catenin in mHSCs was also increased by day 10. mHSCs were treated with TGF-?, Wnt3a, or Wnt5a on day 5, and harvested on day 7. Compared to untreated mHSCs, expression of col1a1 mRNA was increased by TGF-?, and was further increased by Wnt3a and Wnt5a. Furthermore, the increase of axin2 mRNA expression and nuclear localization of ?-catenin confirmed the canonical role of Wnt3a in mHSCs challenged with Wnt3a, but not TGF-? or Wnt5a. Moreover, cultured human HSCs (LX2s) increased col1a1 mRNA expression when pre-treated with TGF-?, then challenged with Wnt3a or Wnt5a. Taken together, these data demonstrate that Wnt3a and Wnt5a can accelerate activation of primary mouse hepatic stellate cells in culture. Future studies should investigate key activators of both canonical and noncanonical Wnt signals as facilitators in HSC activation.
Abs#6. Kupffer Cells and Alcohol: Investigating the role of Mitochondrial C5aR1
Shannon Twardy & Rebecca McCullough
1. Department of Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA. Email: Shannon.twardy@cuanschutz.edu
The innate immune system is an important contributor to the pathogenesis of alcohol-associated liver disease (ALD). Kupffer cells (KC), the resident macrophage of the liver, release pro-inflammatory cytokines (i.e. TNF? and IL-1?) and reactive oxygen species (ROS) upon activation by pathogen associated molecular patterns (PAMPs). Complement, a component innate immunity, contributes to the pathogenesis of ALD; ethanol activates complement in the liver to produce potent anaphylatoxins (C5a and C3a). Mice deficient in the anaphylatoxin receptors C3aR1 and C5aR1 are protected from ethanol-induced liver injury, however the role for surface and intracellular complement receptors has yet to be established. Intracellular complement has been implicated as a modulator of mitochondrial stress responses in macrophages following PAMP activation but the interplay between the complosome and macrophage function in ALD is unknown. We hypothesize that mitochondrial C5aR1 (mtC5aR1) contributes to cellular and mitochondrial-derived ROS (mtROS) production during PAMP activation in ethanol-sensitized KCs. Here, immortalized mouse KCs (ImKCs) were cultured overnight with 50 mM EtOH then stimulated with ethanol and PAMPs (zymosan, PolyinosinicÐpolycytidylic acid, lysophosphatidic acid, and lipopolysaccharide [LPS]) up to 48hours. Proinflammatory markers, complement factors, and cellular and mitochondrial ROS were assessed via qRT-PCR and immunofluorescence in the absence and presence of C5aR1 antagonists with low (PMX205) and high cell permeability (JPE1375). Ethanol increased expression of interleukin-1beta (il-1b( and complement component 3 (c3) mRNA; ethanol in combination with PAMPs increased expression of tumor necrosis factors alpha (tnf-a), il1b, and c3 mRNA (1 hr). Ethanol- and LPS-induced expression of complement component 5 (c5) and c5ar1 mRNA and protein was delayed compared to C3 (24hrs). Ethanol and LPS increased CellROX and MitoSOX fluorescence, which was attenuated by PMX205. JPE1375, but not PMX205, attenuated IL-1b secretion by ImKCs by LPS. Taken together, these data specify distinct roles of surface and mtC5aR1 in ethanol- and LPS-activated ImKCs and may suggest a role for mtC5aR1 in catabolic processes and regulation of NLRP3 inflammasome consistent with complosome findings in other immune cells. While more research is warranted, the complosome may be a novel therapeutic target for progressive liver diseases. This work supported by NIH grants to RLM: R00AA025386, R21ES032529
Abs#7. Reduced toxicity of menadione: the potential role of isotope exchange
Mohsen Ranjbar1, Ahmed R. Muhyi2, Tamara A. Anderson2, Graham Timmins2, Todd A. Thompson2
1. Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, New Mexico 87131
2. Department of Pharmaceutical Sciences, University of New Mexico College of Pharmacy, Albuquerque, New Mexico 87131
Quinone derivatives, like menadione (vitamin K3), are renowned substances in toxicological studies. Menadione, as a quinone with an unsubstituted carbon in the ring exhibits toxicity, in part, through a Michael addition reaction with depletion of beneficial nucleophiles such as glutathione in cells. In addition, menadione may produce toxicity through the production of reactive oxygen species (ROS) via redox cycling. The role of NADPH quinone oxidoreductase 1 (NQO1) as a key enzyme in the metabolism of quinones is noteworthy. The presence of less common isotopes in the structure of xenobiotics has been a thought-provoking topic in toxicology and medicinal chemistry. Our hypothesis is that exchanging oxygens in the structure of menadione with a heavier isotope (such as 18O) could decrease the toxicity of this molecule. Oxygen exchange reaction was performed in the presence of dioxane, high pressure (10 atm), and 120¡C, using 18OH2 as the source of the heavy isotope. A toxicity assay for 16O and 18O-menadione was performed using PC3 human prostate cancer cells. 18O-menadione was shown to produce significantly lower toxicity with an IC50 of 25 ?M for 16O-menadione compared to an IC50 of 40 ?M for 18O-menadione. Our suggestion for this difference is that the presence of 18O would increase the length of the C=O bond on the ring, and the molecule would turn into a better ligand for NQO1 to be metabolized in a less cytotoxic way.
Abs#8. In Vitro Metabolic Study of Sirolimus: Preparation, Isolation, and Identification of the Metabolites
Baharak Davari, Touraj Shokati, Uwe Christians
1. University of Colorado, Anschutz Medical Campus.
Sirolimus (known as rapamycin, SRL) is a triene macrolide lactone isolated from Streptomyces hygroscopicus that has been shown to possess potent immunosuppressant and antiproliferative properties. Due to SRLÕs therapeutic nature, it is critical to understand the human metabolism of the drug. This study focused on the identification of unknown SRL metabolites generated after incubation of human liver microsomes. Previously, 12-hydroxy, 14-hydroxy, 23/24-hydroxy, hydroxy-piperidine, 16-O-desmethyl, and 39-O-desmethyl had been described. Metabolite structures were identified using high-resolution mass spectrometry QTOF, ion trap MSn, and comparison of fragmentation patterns of the metabolites to SRL. Moreover, enzyme kinetic parameters of SRL metabolite formation were investigated. Human liver microsomes mainly hydroxylated and/or demethylated SRL. The structures of the following metabolites were identified:ÊO-demethylated metabolites: 39-O-desmethyl, 16-O-desmethyl and 27-O-desmethyl SRL; hydroxylated metabolites: hydroxy piperidine SRL, 11-hydroxy, 12-hydroxy, 14-hydroxy, 23-hydroxy, 24-hydroxy, 25-hydroxy, 46-hydroxy,49-hydroxy, as well as didemethylated and dihydroxylated metabolites. This study found SRL is metabolized into more than 18 unique metabolites. These metabolites could be structurally identified using ion trap MSnÊand high-resolution mass spectrometry QTOF. Adipokine secretion in the bone niche shifts in response to tungsten exposure in both non-tumor and 4T1 tumor-bearing mice.
Abs#9. Adipokine secretion in the bone niche shifts in response to tungsten exposure in both non-tumor and 4T1 tumor-bearing mice.
Charlotte M. McVeigh1, Cameron Chock1, Sage Templeton1, and Alicia M. Bolt1.
1. University of New Mexico, College of Pharmacy, Department of Pharmaceutical Sciences, Albuquerque, NM, 87131.
Tungsten is classified as an emerging environmental toxicant due to increasing exposure and lack of knowledge about the human health risks. Epidemiological and in vivo studies have demonstrated that exposure to tungsten contributes to the carcinogenic process. However, there is a gap in knowledge in our understanding of how tungsten drives these processes. Due to a cohort of breast cancer patients accidentally exposed to tungsten during intraoperative radiotherapy, our lab is currently investigating the effects of tungsten exposure on breast cancer progression and metastasis. Tungsten is known to accumulate within the bone, creating a site for long-term exposure and toxicity. Breast cancer is also known to metastasize to the bone. We have previously shown using the 4T1 orthotopic breast cancer model that oral tungsten exposure enhances metastasis, osteolysis and myeloid-derived suppressor cells in the bone niche. These findings suggest that tungsten deposition in the bone creates a favorable microenvironment to promote metastasis. Bone marrow adipocytes (BMA) play an important role in breast cancer metastasis to the bone through the secretion of adipokines that drive tumor cells homing, colonization, and growth by changing the microenvironment. Previous research has shown that tungsten exposure in drinking water leads to an increase in adipocytes within the bone marrow. We aimed to evaluate the gene expression of adipokines in bone marrow isolated from both non-tumor and 4T1 tumor-bearing mice following tungsten exposure. Tungsten exposure shifted the adipokine profile in the bone niche, in both non-tumor and 4T1 tumor-bearing mice. Interestingly, the adipokines that were elevated following tungsten exposure in each group were different. These results suggest that adipocytes could be important mediators driving tungsten-enhanced breast cancer metastasis to the bone. Future work will focus on investigating how changes in bone adipocytes following tungsten exposure contribute to this process.
Abs#10. Identifying molecular biomarkers following tungsten particulate exposure in human lung fibroblast cells.
Delfina Lozoya1, Sage Templeton1, Alicia M. Bolt1.
1. University of New Mexico, College of Pharmacy, Department of Pharmaceutical Sciences1, Department of Pharmacy Practice & Administrative Sciences2, Albuquerque, NM, 87131.
Epidemiological studies have established a link between exposure to tungsten and increased risk of developing cardiovascular disease and stroke. However, little is known about how tungsten affects cardiac function or the risk of developing cardiovascular disease. We have previously shown that acute inhalation of tungsten particulates leads to alterations in cardiac function including decreased cardiac output, increased work required by the atria to fill the heart (AÕ), and a decreasing trend in EÕ/AÕ ratio, an indictor of diastolic relaxation. Importantly, expression analysis of heart tissue revealed molecular changes consistent with these biological outcomes, including increased expression of pro-inflammatory, cardiac remodeling and fibrosis, and oxidative stress genes. Tungsten deposition in lung and heart tissue indicated that tungsten accumulates in the lungs, but not the heart following inhalation exposure suggesting alterations in the lungs are driving the changes in the heart observed. Previous studies have shown that the lung microenvironment is altered following tungsten particulate exposure including increased inflammation, fibroblast activation, and metabolomic changes. This study focused on in vitro tungsten particulate treatment of HFL-1 human fibroblast cells in order to identify molecular changes in the lungs that could travel into the systemic circulation to alter cardiac function. HFL-1 cells were exposed to tungsten particulate concentrations: 5, 10, and 25 ug/mL for three days. Gene expression of the following biomarkers were analyzed: pro-inflammatory cytokines Il-1? and Il-6, markers of remodeling and fibrosis Tgf-?1 and Acta-2, and markers of oxidative stress Hif-1? and Hmox-1. Tungsten particulate exposure increased the expression of all 6 genes following exposure, however data were only statistically significant for Il-6 and Il-1?. Interestingly, some of the responses were not dose dependent, which makes analyzing the results challenging. These data have identified potential biomarkers in the lungs following tungsten particulate exposure that could be systemic drivers of altered cardiac function. Current work is focused on validating these biomarkers in in vivo models.
Abs#11. Do extracellular vesicles from canines with Canine cognitive dysfunction cause neuroinflammation?
Sean Boland1, Amelia Hines1, Arielle Hay2,Stephanie McGrath3,and Julie A. Moreno1
1. Department of Environmental and Radiological Health Sciences
2. Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523
3. Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA
Extracellular vesicles (EVs) are shown to play a pivotal role in cell-cell signaling, cellular homeostasis, and waste deposition. However, they are beginning to be explored as possible routes for initiation and/or progression of neurodegenerative diseases, including AlzheimerÕs disease (AD) and other age associated diseases. EVs cargo include but are not limited to proteins, nucleic acids, and signaling molecules. It has been shown that EVs from normal healthy cells contain cargo that possess neuroprotective properties. Conversely, unhealthy cells or cells under neuroinflammatory conditions contain cargo that can be damaging to neurons and can lead to neuronal death. Astrocytes, the cells that maintain neuronal health, can express pro-inflammatory (A1) or anti-inflammatory (A2) phenotypes and the contents of their EVs will be vastly different. It has been shown that astrocytes stimulated with pro- inflammatory cytokines Tnf-? and IL-1b, secrete EVs with less neuroprotective properties and secrete more EVs with cargo that prolongs neuroinflammation. AD models mainly use rodent models. However, aging canines have a naturally occurring disease that mimics AD, canine cognitive dysfunction (CCD) syndrome. We therefore are investigating if EVs from an aged canine can stimulate human astrocytes to a pro-inflammatory (A1) phenotype. Our hypothesize is that EVs from aged canines will contain cargo that is neuroinflammatory, compared to EVs from aged match non-CCD positive canines. Our read-outs for this will be quantitative real-time PCR and ELISAs to determine the degree of A1 phenotype.
Abs#12. Identifying molecular mechanisms associated with particulate matter (PM2.5) exposure and osteoarthritis
Afzali, Maryam F., Schenkel, Alan and Santangelo, Kelly S.
1. Department of Microbiology, Immunology & Pathology, Colorado State University, Fort Collins, CO
Osteoarthritis (OA) is complex and multifactorial, with many interconnected cellular pathways influencing disease onset and progression. Epidemiological studies have suggested that environmental factors such as pollution may contribute to an increased risk of OA. Recently, investigators have reported an increased prevalence of pulmonary disease in relationship to PM2.5 exposure and made a link to musculosketelal conditions. While some work has investigated PM in animals models of induced post-traumatic OA or rheumatoid arthritis, there are no studies to date that explore the effect of PM2.5 exposure in an age-related, naturally-occurring model of OA. We postulated that chronic exposure to PM2.5 accelerates the pathogenesis of primary OA via increased inflammation, indirectly and/or directly. Indirect interactions, whereby lung inflammation incites a cascade of cytokines into the systemic circulation, may then influence joint homeostasis. Alternatively, PM2.5 may also be directly influencing joint health through accumulation in these tissues. We began with in vitro work to establish that cell viability of primary articular chondrocytes from an animal model of primary OA decreases significantly after 48 and 72 hoursof PM2.5 exposure (100 ?g/ml). Further, primary chondrocytes exposed to PM2.5 for 72 hours significantly increased reactive oxygen species (ROS) production when compared to control primary chondrocytes. Similarly, Annexin V staining corroborated a significant increase in apoptotic chondrocytes exposed to PM2.5. Cumulatively, this in vitro work provided the foundation that primary chondrocytes can undergo oxidative stress and programmed cell death with PM2.5. To evaluate the in vivo effect of PM2.5, OA prone Dunkin-Hartley guinea pigs inhaled fresh air (FA) or PM2.5 for 1 hour for 7 consecutive days. Significant increases in MCP-1, PGE2, and C3 (key inflammatory mediators associated with joint degeneration) were present in both serum and joint fluid. Normalized mRNA counts from guinea pig articular cartilage and mensicus revealed that key signal transduction pathway molecules (MAPK, JAK2 and STAT2) were significantly increased. Pro-apoptotic (BAX, BAK, CASP8 and CASP9) molecules were increased, while anti-apoptotic (BCL-2 and BAD) markers were significantly decreased. in PM2.5 exposed tissue. Collectively, this data suggests that animals display oxidative stress, as well as systemic and local inflammation, when exposed to PM2.5.
Abs#13. The investigation of alcohol-induced lysine N-acetylation and the roles of cysteine proximity as a driver of hepatic proteome dysregulation
Courtney D. McGinnis, Peter S. Harris, John Marentette, Cole Michel, Laura Saba, Richard Reisdorph, James Roede, Kristofer S. Fritz
University of Colorado Anschutz Medical Campus
Alcohol-associated liver disease (ALD) is a leading cause of preventable mortality worldwide causing about 5.9% of deaths every year. The onset of ALD begins with steatosis affecting 90-100% of patients that overconsume alcohol (>40g of alcohol per day) and 10-35% of that population will develop steatohepatitis. The liver is the primary site of the oxidative metabolism of alcohol and is significantly impacted by these products (acetaldehyde and acetate), negatively impacting hepatic biochemistry. Two significant regulatory consequences are the induction of lysine acetylation, a metabolically induced posttranslational modification (PTM), and changes to cysteine signaling. Here, we examined the impact of chronic alcohol metabolism on hepatic lysine acetylation and cysteine redox through quantitative analysis via acetyl-IP and redox click chemistry enrichments followed nHPLC-MS/MS. Interestingly, redox regulating enzymes were found to be hyperacetylated during alcohol metabolism and contribute to an altered redox state of hepatic protein thiols (cysteine) on these same proteins. These findings are supported by a spatial association between lysine and cysteine amino acids. Lysine acetylation is amplified when cysteine residues are within 15 , referred to as a cysteine-lysine pair (CysLys). This alcohol-induced increase in N-acetylation is partially due to the acetylation of the more reactive cysteine sulfide followed by transfer to a nearby lysine amine, resulting in an S-->N acetyl transfer reaction. Our findings support mechanisms of S-->N acetyl transfer reactions, where proteins found to be acetylated as a result of alcohol metabolism are more likely to contain a perturbed thiol group(s). Additionally, our acetylomic and redox proteomic datasets are significantly enriched for CysLys pairs reported to be evolutionarily conserved. An integrative bioinformatics approach using pathway analysis software suites was employed to gain insight regarding how murine hepatic protein pathways are altered by alcohol toxicity. Cysteine and lysine proximity impacts proteins that are involved in redox reactions, such as oxidoreductases. Additionally, our results demonstrate that alcohol metabolism directs redox signaling across cellular processes such as the citric acid cycle, electron transport chain, beta-oxidation, and sirtuin signaling. Further research is needed to determine how CysLys acetylation dynamics impact individual protein activity to elucidate how these critical pathways are disturbed.
Abs#14. Per- and Polyfluoroalkyl Substances (PFAS) Inhibit Neonatal Cytochrome P450 CYP3A7
Michaela Hvizdak, Sylvie E. Kandel, and Jed N. Lampe
University of Colorado Anschutz Medical Campus
Per- and polyfluoroalkyl substances (PFAS) are a chemical class of highly fluorinated aliphatic compounds known for their oleophobic and hydrophobic qualities as well as their physicochemical stability. These characteristics make them popular for use in consumer products, including clothing, carpeting, cookware, and food and toy packaging. PFAS environmental persistence in drinking water allows for acute exposure to humans and subsequent bioaccumulation of the compounds in the liver and lung tissue. Prenatal PFAS exposure has been associated with lowered birth weight, premature birth, and developmental defects including cranio-facial abnormalities. The neonatal cytochrome P450 (CYP450) enzyme CYP3A7 is responsible for facilitating a variety of reactions essential for proper fetal development in humans. In addition to drug metabolism, CYP3A7 is responsible for metabolizing endogenous ligands including the steroid precursor dehydroepiandrosterone 3-sulfate (DHEA-S), along with the morphogen all-trans-retinoic acid (atRA) in the developing human liver. Inhibition of CYP3A7, and subsequent interference with estriol synthesis, as well as atRA clearance is known to result in similar effects associated with prenatal PFAS exposure including lowered birth weight, premature birth, and developmental defects. We hypothesized that PFAS compounds bind to the neonatal CYP3A7 enzyme and inhibit metabolism of DHEA-S and atRA. We implemented a series of binding studies using spectral characterization of six PFAS compounds (PFOA, PFOS, GenX, PFNA, PFNS, and PFHxS), and evaluated their interactions with recombinant CYP3A7. In addition, we measured the levels of inhibition of the oxidation of dibenzylfluorescein (DBF), a fluorescent probe, in a CYP3A7 activity assay. IC50 values were obtained from compounds that showed significant inhibition of CYP3A7. Our data demonstrate that of the six PFAS tested, PFOA, PFOS, PFNA, and PFHxS bind to CYP3A7. Furthermore, PFOS and PFNA were shown to significantly inhibit CYP3A7 at biologically relevant concentrations. These results not only point to a possible explanation for the developmental defects associated with prenatal PFAS exposure, but suggest potential for PFAS to bind to and inhibit other CYP450 enzymes involved in fetal development as well.
Abs#15. Progressive Gliosis Caused by Systemic Tuberculosis Disease
Amanda S Latham1, Charlize Geer1,2, Isla Anderson1,2, David F Ackart2, Amelia Day Hines1, Brendan Podell2, Jessica Elf1, Randall J Basaraba2, and Julie A Moreno1
1. Department of Environmental Health and Radiological Sciences, Colorado State University
2. Department of Microbiology, Immunology, and Pathology, Colorado State University
Approximately one-quarter of the worldÕs population is latently infected with Mycobacterium tuberculosis (Mtb), the causal agent of Tuberculosis (TB), with 10 million new cases of active TB annually. Co-infection with human immunodeficiency virus (HIV) and TB exacerbates cognitive deficits compared to HIV infection alone. Additionally, epidemiologic studies demonstrate that pulmonary tuberculosis causes memory loss and predisposes individuals to catastrophic neurodegenerative diseases. This includes ParkinsonÕs Disease (PD) and dementia, which affect approximately 60 million people worldwide. That number is estimated to dramatically increase over time due to rising aging populations and the effects of infectious diseases, such as TB. Through studies examining the neurological effects of low-dose Mtb exposure by aerosol, we identify pathological markers of inflammation in the central nervous system (CNS) of outbred, Dunkin Hartley guinea pigs. This includes migration and proliferation of microglia followed by activation of astrocytes in multiple brain regions. Interestingly, these glial effects, or gliosis, are present despite the absence of detectible bacteria in the brains of infected animals. These results are not only found in guinea pigs, a well-established model of pathological TB progression, but are also evident in Mtb-infected CD-1 mice and non-human primates. Through this data, we aim to fully characterize the inflammation and subsequent neurotoxicity that accompanies systemic TB disease. These studies will allow us to obtain a better understanding of the disease progression in the hopes of alleviating permanent cognitive impairments in human patients.
Abs#16. High-content imaging analysis for the testicular toxicity prediction using an 87-Compound library in a three-dimensional testicular cell co-culture model
Lei Yin2, Zoey Hsuan Hsiao2, Xiaozhong (John) Yu1,
1. College of Nursing, University of New Mexico, Albuquerque, NM 87131
2. Reprotox Biotech LLC, 800 Bradbury, Drive, SE, Science &Technology Park, Albuquerque, NM, 87106
3. Correspondence Address: xiyu@salud.unm.edu, lei@ivtox.com
This study aims to develop a novel in vitro alternative testing platform for male reproductive toxicity to support the principle of reducing, replacing, and refining (3R) and reducing the time and costs associated with reproductive toxicity testing. In our previous study, we developed an in vitro three-dimensional (3D) testicular cell co-culture model (3D-Testis) and observed a strong correlation between the in vitro IC50 and in vivo rLOAEL with 32 tested compounds. This study integrated the 3D-Testis model with a High-content image-based single-cell phenotypic analysis and tested an 87-compound library to validate this in vitro model. This 87-compound library provided by the National Toxicology Program (NTP) included known and suspected toxicants such as flame retardants, drugs, industrial chemicals, polycyclic aromatic hydrocarbons (PAHs), pesticides as well as negative controls. The co-cultured cells were treated with 87 compounds in a wide range of doses (1-100?µM), and multi-parametric high-content analysis (HCA) was conducted. High-content assays included multiple endpoints of adverse outcome pathways, including nuclear morphology, DNA synthesis, DNA damage, and cytoskeleton structure. Compounds were ranked by potency on individual endpoints and selectivity of adverse outcome signaling pathways for hazard prioritization. None of the presumptive negative controls nor vehicle controls were shown toxicity in the model. Dose-dependent changes of these known reproductive toxicants were observed. Furthermore, we found differential toxicity profiles were potentially associated with their unique mechanisms of toxicity. This study not only validates the efficiency of this in vitro model but also tests the robustness and relevance of the model system with multiple endpoints associated with adverse outcome pathways of the male reproductive system. (Supported by NIEHS R44 ES027374 and R43 ES031890)
Abs#17. Plasma biomarkers as a diagnostic tool for canine cognitive dysfunction
Amelia Day Hines, Sean Boland, Breonna Kusick, Stephanie McGrath, and Julie Moreno
Canines, like humans, develop many features of human aging, including cognitive decline and neuropathologies including neuroinflammation and accumulation of misfolded proteins, making them an excellent translational model for diseases like AlzheimerÕs and other human dementias. Currently, the only antemortem diagnostic available for canine cognitive dysfunction (CCD) is the use of magnetic resonance imaging (MRI) which primarily identifies other causes of cognitive decline including brain infections and cancer. Development of antemortem diagnostics using both blood and cerebrospinal fluid (CSF) samples is vital to the early diagnosis of aged canines. The challenges posed by this however is low concentrations of known biomarkers, such as misfolded amyloid-beta (A?), hyperphosphorylation of tau (P-tau), inflammatory marker glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL) in aged canines. By extracting extracellular vesicles to detect A?, P-tau, GFAP and NfL, we have been able to correlate changes in these markers to pathological changes in the brain tissue of the same canines. Use of these sensitive diagnostic tools by veterinarians would provide an enormously beneficial tool for the early and accurate diagnosis of CCD. The translational ability of the canine model for human dementia and AlzheimerÕs disease allows for exciting research opportunities to move forward diagnostic testing in humans.
Abs#18. Immune dysregulationÊassociated with metal exposure on Navajo Nation
T. Anderson1, N. Thompson1, L. Luo2, E. Dashner Titus3, L. Hudson3 and D. MacKenzie1
1. University of New Mexico Health Sciences Center, Community Environmental Health Program, College of Pharmacy, Albuquerque, NM
2. Department of Internal Medicine and UNM Comprehensive Cancer Center, University of New Mexico, Albuquerque, NM
3. University of New Mexico Health Sciences Center, College of Pharmacy, Albuquerque, NM
Many tribal populations are characterized by health disparities including higher rates of infection, metabolic syndrome, and cancer Ð all of which are mediated by the immune system. Members of the Navajo Nation have suffered chronic low-level exposure to metal mixtures from uranium mine wastes for decades. We suspect that such metal exposures lead to adverse health effects via their modulation of immune system function. We examined the relationships between 9 key metal exposures (in blood and urine) with 11 circulating biomarkers (cytokines and CRP in serum) in 160 pregnant Navajo women. Biomonitored levels of uranium and arsenic species were considerably higher in participants than NHANES averages. Further, each metal exposure except Hg, was associated with a unique set of cytokines. Where arsenic species were generally immunosuppressive (decreased cellular and humoral stimulating cytokines), uranium appeared to encourage the development of autoimmunity (higher IL-17). In our Thinking Zinc clinical trial we are investigating whether zinc status can modulate cytokine alterations associated with uranium and arsenic exposures in a non-pregnant adult population based on the interaction of these metals with key zinc-binding proteins. Overall, our results are preliminary to suggest that environmental metal exposures modulate immune status in a potentially interactive fashion.
Abs#19. High Dose of Aerosolized Mycobacterium tuberculosis Induces Neurotoxicity and Gliosis in CNS of Guinea Pigs
Charlize E Geer1, Amanda Latham2, David F Ackart1, Amelia Day Hines1, Randall J Basaraba1, Julie A Moreno2
1. Departments of Microbiology, Immunology and Pathology1 and Environmental Radiological Health Sciences2, Colorado State University
Tuberculosis (TB) remains one of the most devastating infectious diseases worldwide, with tuberculosis meningitis (TBM) being the more deadly form. TBM occurs when Mycobacterium tuberculosis (Mtb) crosses the blood brain barrier (BBB) and causes inflammation of the meninges and brain damage. Additionally, central nervous system (CNS) damage can occur when the intensely immunogenic response to peripheral Mtb infection of the lungs reaches the brain. Currently, the most common model for TBM utilizes rabbits infected with Mtb by subarachnoid inoculation. While effective, this model does not reflect natural exposure to Mtb nor disease progression comparative to that of human patients. We have proposed a more effective method of studying TBM through the use of guinea pigs, which show disease pathology and symptoms similar to human TB infection, given a high dose of Mtb by aerosol exposure. In this study, young Dunkin Hartley guinea pigs were infected with high doses of one of two Mtb strains, either the common laboratory strain H37Rv or the hypervirulent strain HN878. Animals were terminated at 15 dpi and then various staining techniques, including immunohistochemistry, were performed to evaluate neurotoxicity. Mtb induced lesions within the brain, and there were increased activated microglia at the sites of damage. Although no Mtb colonies were cultured from brain homogenate, our preliminary findings demonstrate that aerosolized infection using high dose and a high virulent strain of Mtb in guinea pigs could serve as a more accurate model to study TBM, rather than directly injecting Mtb into the CNS.
Abs#20. Histopathological and Immunological Characterization of Repeated Ozone Exposure in the Healthy and Susceptible Lung
J Nguyen, C Deering-Rice, C Reilly, and A Venosa
1. Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT
Exposure to the ubiquitous air pollutant ozone (O3) is linked to the morbidity and mortality of millions worldwide. Acute O3 exposure is known to produce immediate and reversible epithelial damage and myeloid dominant inflammation in the healthy lung. By comparison, these responses are much less defined in the context of repeated exposure of at-risk populations, including those presenting genetic predisposition to pulmonary disease. We therefore set to examine the impact of repeated O3 exposure (3h, 0.8ppm, 4x days) in healthy and predisposed cohorts. The isoleucine-to-threonine substitution in the epithelial type-2 cell-specific Surfactant Protein C protein (SP-CI73T) was used as a clinically relevant model of epithelial driven stress linked to fibrogenic remodeling. In vivo lung mechanics, bronchoalveolar lavage, as well as lung tissues for histological, and flow cytometric analysis were performed 24 and 72 h post last exposure. FlexiVent analysis showed divergent responses at 24 h, with significant differences in P-V loop areas and elastance. These results were consistent with histological evidence of septal remodeling and perivascular edema. Cell counts revealed limited inflammatory cell influx in the bronchoalveolar space, while flow cytometry of lung digests identified progressive myeloid-to-lymphoid shift, maximal 72 h post exposure. Healthy cohorts displayed significant increases in CD3 lymphocytes, natural killer (NK) cells, and B220+ B cells after exposure. By comparison, SP-CI73T mice was associated with recruitment of Ly6C+ monocytes and mannose receptor-expressing (CD206) monocyte-derived macrophages, at 24 h. In support of this evidence, histochemical analysis also showed clusters of CD206+ macrophages within areas of septal remodeling. Furthermore, increase in B220+ B cell were limited, and NK presence was nearly ablated in control and O3 conditions. Taken together, our results demonstrate that repeated ambient ozone exposure generates an extensive phenotype, with distinct functional and inflammatory responses between healthy and mutant/null cohorts. This work provides a foundational framework to study complex exposure paradigms across the spectrum of susceptibility consistent with human disease Grant support: NIEHS R01ES032553
Abs#21. Detection of Transgene Location in the CYP2A13/2B6/2F1-transgenic Mouse Model using Optical Genome Mapping Technology
Xinxin Ding, John Han, Laura Van Winkle, and Qing-Yu Zhang
1. Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ 85721 (XD, JH, QZ);
2. Center for Health and the Environment, UC Davis, Davis, CA 95616 (L.V.)
Most transgenic mouse models are generated through random integration of the transgene. The location of the transgene, which provides valuable information for assessing potential effects of the transgenesis on the host and for designing genotyping protocols that can amplify across the integration site, is challenging to identify. Here, we report the successful utility of the optical genome mapping technology to identify the transgene insertion site in a CYP2A13/2B6/2F1-transgenic mouse model, which produces three human P450 enzymes (CYP2A13, CYP2B6, and CYP2F1) that are encoded by neighboring genes on human Chr.19. These enzymes metabolize many drugs, respiratory toxicants, and chemical carcinogens. Initial efforts to identify candidate insertion sites by whole genome sequencing was unsuccessful. Subsequent utility of the optical genome mapping approach, which compares genome-wide marker distribution between the transgenic mouse genome and a reference mouse (GRCm38) or human (GRCh38) genome, localized the insertion site to mouse Chr.14, between two marker positions at 4451324 bp and 4485032 bp. A transgene-mouse genome junction sequence was further identified through long-PCR amplification and DNA sequencing at GRCm38 Chr14:4484726. The transgene insertion (~2.4 Mbp) contained 5-7 copies of the human transgenes, which replaced a 26.9Ð33.4 kbp mouse genomic region, including exons 1-4 of Gm3182, a predicted and highly redundant gene. Finally, the sequencing results enabled the design of a new genotyping protocol that can distinguish between hemizygous and homozygous CYP2A13/2B6/2F1-transgenic mice. Our success in applying the optical genome mapping technology for identification of transgene insertion sites should encourage others to do the same for other transgenic models generated through random integration, including most of the currently available human P450 transgenic mouse models. (Supported in part by NIH grants CA092596 and ES020867)
Abs#22. Downregulation of airway mucins by low-dose arsenicals
Shelby Herrick, Fangwei Liu , Weifeng Liang , Lingxiang Zhu, R Clark Lantz, Yin Chen
1. Department of Pharmacology and Toxicology, R. Ken Coit College of Pharmacy, University of Arizona, Tucson, AZ 85718
Arsenic is a ubiquitous environmental toxicant, found in high concentrations worldwide. Although abundant research has dealt with arsenic-induced cancers, studies on mechanisms of non-malignant lung diseases have not been complete. In addition, decades of research have mostly concentrated on high-dose arsenic exposure, which has very limited use in modeling the biological effects of today's low-dose exposures. Indeed, accumulated evidence has shown that low-dose arsenic exposure (i.e. ²100 ppb) may also alter lung homeostasis by causing host susceptibility to viral infection. However, the underlying mechanism of this alteration is unknown. In this study, we found that low-dose sodium arsenite (As (III)) repressed major airway mucins-MUC5AC and MUC5B at both mRNA and protein levels. We further demonstrated that this repression was not caused by cellular toxicity or mediated by the reduction of a common mucin-inducing pathway-EGFR. Other established mucin activators- dsRNA, IL1? or IL17 were not able to override As (III)-induced mucin repression. Interestingly, the suppressing effect of As (III) appeared to be partially reversible, and supplementation of all trans retinoic acid (t-RA) doses dependently restored mucin gene expression. Further analyses indicated that As (III) treatment significantly reduced the protein level of retinoic acid receptors (RAR?, ? and RXR?) as well as RARE promoter reporter activity. Therefore, our study fills in an important knowledge gap in the field of low-dose arsenic exposure. The interference of RA signaling, and mucin gene expression may be important pathogenic factors in low-dose arsenic induced lung toxicity.
Abs#23. Normal human kidney cell response to environmental metals.
Jodi R. Schilz1, Erica J. Dashner-Titus2, Karen Simmons2, Laurie G. Hudson2
1. University of New Mexico, Division of Physical Therapy
2. Department of Pharmaceutical Sciences, Albuquerque, NM
Chronic metal exposure from abandoned mines and waste piles pose unknown health risks to populations living in their proximity. Metals elevated in environmental or biological samples from people living near mine and milling sites in New Mexico include arsenic (As), cadmium (Cd), uranium (U) and vanadium (V). ÊMetals can enter the body through inhalation of dust, contact with skin and/or consumption of food and water and are associated with increased incidence of chronic disease, including that of the kidney. To address the potential effect of these metals on primary kidney cells, we used RNA sequencing methods to determine the effect of acute, sub lethal exposure to As, U, Cd, and V. PCA plot analysis of differentially expressed genes shows separation of U, V and As on the PC1 (38.49%) with only Cd on the PC2 (15.95%). As treatment showed the greatest number of significantly expressed genes compared to control, followed by Cd, V and U. REACTOME enrichment analysis revealed the ÒCellular response to external stimuliÓ pathway was significantly enriched with As and Cd treated cells. This pathway is a signal transduction super pathway that includes macroautophagy, response to metal ions and stress response to ROS, hypoxia and heat. qRT-PCR analysis verified enhanced gene transcription of oxidative stress genes, HMOX1 and NQO1, in response to metal, time and dose exposure. Additionally, GO enrichment analysis revealed enhanced enrichment of pathways involved in autophagy and autophagic mechanisms with As and U exposure. Cells stained for LC3 in the presence of As, U, Cd for 24 hours showed increased fluorescence. The results demonstrate the need to investigate metals at environmentally relevant concentrations to better understand the toxicological impact of these mixtures on normal human cells along the major exposure routes.
Abs#24. The Drinking Water and Swimming Pool Disinfectant Trichloroisocyanuric Acid Causes Chlorination Stress Enhancing Solar UV-Induced Inflammatory Gene Expression in AP-1 Transgenic SKH-1 Luciferase Reporter Mouse Skin
Jeremy A Snell 1,2, Prajakta Vaishampayan2,3, Sally E Dickinson2,3, Jana Jandova1,2, Georg T Wondrak1,2,
1. Department of Pharmacology and Toxicology, R.K. Coit College of Pharmacy, University of Arizona, Tucson, AZ;
2. UA Cancer Center, University of Arizona, Tucson, AZ;
3. Department of Medical Pharmacology, University of Arizona, Tucson, AZ, USA.
Freshwater sanitation and disinfection using a variety of chemical entities as chlorination agents is an essential public health intervention ensuring water safety for populations at a global scale. Recently, we have published our observation that the small molecule oxidant, innate immune factor and chlorination agent HOCl antagonize inflammation and photocarcinogenesis in murine skin exposed topically to environmentally relevant concentrations of HOCl. Chlorinated isocyanuric acid derivatives (including the chloramines trichloroisocyanuric acid [TCIC] and dichloroisocyanuric acid [DCIC]) are used worldwide as alternate chlorination agents serving as HOCl precursor and stabilizer compounds ensuring sustained release in aqueous environments including public water systems, recreational pools and residential hot tubs. Here, for the first time, we have examined the cutaneous TCIC-induced transcriptional stress response (in both an organotypic epidermal model and in AP-1 luciferase reporter SKH-1 mouse skin), also examining molecular consequences of subsequent treatment with solar ultraviolet (UV) radiation. Taken together, our findings indicate that cutaneous delivery of TCIC significantly enhances UV-induced inflammation (as profiled at the gene expression level), suggesting a heretofore unrecognized potential to exacerbate UV-induced functional and structural cutaneous changes. These observations deserve further molecular investigations in the context of TCIC-based freshwater disinfection with health implications for populations worldwide. (Supported in part by NIEHS; 1R21ES029579; ES006694; ES007091)
Abs#25. Arsenic selectively alters mutational patterns of UVR exposure
Rachel M. Speer1, Shuvro Nandi2-4, Xixi Zhou1, Yan Guo5, Laurie G. Hudson1, Ludmil B. Alexandrov2-4, Ke Jian Liu1*
1. Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico, Albuquerque, New Mexico, USA.
2. Department of Cellular and Molecular Medicine, UC San Diego, La Jolla, CA, 92093, USA
3. Department of Bioengineering, UC San Diego, La Jolla, CA, 92093, USA
4. Moores Cancer Center, UC San Diego, La Jolla, CA, 92037, USA
5. Department of Internal Medicine, Division of Molecular Medicine, University of New Mexico, Albuquerque, New Mexico, USA.
Arsenic is a global health concern and environmental pollutant. Many studies focus on arsenic as a potent carcinogen targeting the lung, bladder, kidney, and skin, while fewer studies have evaluated arsenic as a co-carcinogen. Although the EPA and WHO set drinking water limits for arsenic (a main source of exposure) to 10 ppb, this limit was based on single factor exposures and does not fully consider arsenic as a co-carcinogen. Arsenic acts as a co-carcinogen at concentrations below the current drinking water limit. Arsenic enhances UVR skin cancer, however, the mechanisms are not fully understood. One proposed mechanism of arsenic co-carcinogenesis involves its interference with the nucleotide excision repair (NER) pathway, which is responsible for repairing cyclobutene-pyrimidine dimers (CPDs), a type of UVR-induced DNA damage known to result in mutations found in UVR skin cancers. In this study a human skin cell model was used to investigate arsenic-altered mutation patterns of UVR exposure. Arsenic alone did not induce mutations, but significantly increased UVR associated mutations. Arsenic also selectively altered the spectra of known UVR mutational signatures. These findings suggest arsenic is selective in altering mutational patterns of UVR co-exposure revealing mechanistic insight on arsenic as a co-carcinogen.
Abs#26. Inhibition of inflammatory pathways prevent prion induced neuroinflammation and progression of disease
Sydney J. Risen1, Sean Boland1, Sadhana Sharma3, Grace Weismann1, Payton Shirley1, Amelia Hines1, Arielle D. Hay1, Stephanie McGrath2, Anushree Chatterjee3, Prashant Nagpal3 and Julie A. Moreno1
1. Department of Environmental and Radiological Health Sciences, and Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA
2. Sachi Bioworks Inc., Colorado Technology Center, Louisville, CO, USA
Neuroinflammation is a key factor in the development of neurodegenerative diseases, including prion diseases, AlzheimerÕs, ParkinsonÕs, and Multiple Sclerosis. Currently, there are little to no effective treatments to halt the progression of neurodegeneration. Similar pathogenesis occurs in all neurodegenerative diseases, including neuroinflammation, accumulation of misfolded proteins, loss of synapses, and cognitive/behavioral deficits. Neuroinflammation may be the result of numerous causes including the accumulation of aggregated proteins and cytokine release from injured glial cells. However, if inflammatory pathways are inhibited, it may be possible to slow the progression of disease. In a prion mouse model, novel Nanoligomersª (Sachi Bioworks) were used to inhibit known inflammatory pathways activated during prion disease. The lead immunotherapy targets were identified through downregulation of several proinflammatory cytokines (e.g., Interleukin-1? or IL-1?, tumor necrosis factor-alpha or TNF-?, TNF receptor 1 or TNF-R1, Interleukin 6 or IL-6), inflammasomes (e.g., NLRP1, NLRP3), key transcription factors (e.g., nuclear factor kappa-B or NF-??) and their combinations, as upstream regulators, and canonical pathway targets, to identify and validate the best-in-class treatment. [SB1]Mice were treated twice a week with the lead neurotherapeutic Nanoligomer SB_NI_112, via two routes of exposure, intranasal and intraperitoneal from 10 weeks of post infection (wpi) to the mice succumbed to the disease. Monitoring behavior and pathogenic changes throughout the disease progression we identified significant decreases in prion induced behavioral deficits, glial inflammation, spongiotic change and neuronal degradation in the brain. We are continuing to assess the treatment of neurotoxicity with these novel nanoligomers in other neurodegenerative mouse models. This work is also being translated into a natural occurring neurodegenerative disease, canine cognitive decline syndrome.
Abs#27. 5-HT3 Antagonist Antiemetic Drugs Alter Cisplatin Exposure and Risk of Kidney Function Decline
Lauren E. Thompson, BS,1 Xia Wen, PhD,3,4 Justine Jorgensen, BS,5 Jordan N. Palan, BS,1 Cathleen L. Doherty, PhD,3 Brian T. Buckley, PhD,3 Edgar A. Jaimes, MD,5 Lauren M. Aleksunes, PharmD, PhD,3,4 Melanie S. Joy, PharmD, PhD1,2
1. University of Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, CO.
2. Division of Renal Diseases, Aurora, CO.
3. Rutgers University, Environmental and Occupational Health Sciences Institute
4. Ernest Mario School of Pharmacy Department of Pharmacology and Toxicology, Piscataway, NJ.
5. Memorial Sloan Kettering Cancer Center, Department of Medicine, New York, NY.
Background: Cisplatin, a common chemotherapeutic, causes acute kidney injury (AKI) in up to one-third of patients. Previous reports have indicated that maximum plasma concentration (Cmax) or area under the plasma concentration vs. time curve (AUC) of platinum (Pt) increase risk of AKI. Ondansetron, a commonly co-prescribed 5-HT3 antagonist antiemetic drug in patients receiving cisplatin-containing chemotherapy, has been associated with enhanced risk of AKI in rodents and retrospective clinical studies. However, to date, there has been no prospective evaluation of AKI risk in patients randomized to different 5-HT3 antagonist drugs.
Methods: As part of study NCT03817970, an initial group of patients (n=16) undergoing their first or second round of cisplatin chemotherapy (³25 mg/m2) were prospectively randomized to one of three 5-HT3 antagonist antiemetic drugs (ondansetron 8 mg p.o., granisetron 2 mg p.o., or palonosetron 0.25 mg i.v.). Total platinum plasma concentrations were quantified using inductively coupled plasma mass spectrometry (ICP/MS) with LLOQ of 1.0 ng/mL. Noncompartmental pharmacokinetic (PK) analysis of total platinum was performed using Certara Phoenixª. Parameters were compared based on 5-HT3 antagonist treatment using one-way ANOVAs with Tukey-Kramer post-hoc tests using R.
Results: 5-HT3 antagonist antiemetic prescription significantly altered exposure to total platinum as evidenced by increases in plasma Cmax (p=0.0167) and AUC (p=0.0034), both normalized by dose of cisplatin, in patients receiving ondansetron. Additionally, ondansetron-treated patients exhibited decreases in estimated glomerular filtration rate (eGFR) or kidney function as compared to granisetron and palonosetron treatment.
Conclusion: 5-HT3 antagonist antiemetic prescription with ondansetron increased exposure to platinum and decreased eGFR. These results suggest that antiemetic treatment with granisetron or palonosetron may be preferred over ondansetron to avoid an increased risk of nephrotoxicity in cisplatin-treated cancer patients.
Abs#28. Disruption of Cytokinesis and Cell Cycle Progression in Bisphenol AF Ð Induced Aneuploidy Multinucleation in the Male Germ Cells
Chelin Hu1, Hsuan Hsiao2, Lei Yin2, Xiaozhong Yu1
1. College of Nursing, University of New Mexico
2. ReproTox Biotech, Albuquerque, NM 87131
Increasing exposure of bisphenol A (BPA), defined as an environmental disrupting chemical, has been shown to be critically associated with cancers, metabolic diseases, neurodegenerative diseases, and dysregulation of male and female reproductive physiology such as infertility. Amongst those eager alternatives to replace BPA in the industry, bisphenol AF (BPAF) is one of the most toxic in reproductive physiology in animals. Previously, we demonstrated that treatment of BPAF resulted increased cell death and unique multinucleations in cultured mouse spermatogonial cells. Compared with other BPA analogs and phthalate compounds, BPAF exhibited a multi-spectral cytotoxicity against all three major testicular cell types in culture. The goal of this study is to understand the mechanisms of BPAF-induced aneuploidy multinucleated gonocytes (MNG). We examined the role of small GTPases, major regulators of the cytokinetic processes, by co-treatment of a selective group of GTPase inhibitors to RhoA and Rac1/Cdc42 alike with BPAF in the mouse spermatogonial C18-4 cells. Single cell-based high-content image analysis (HCA) revealed that 5 µM BPAF resulted in 40% of cell loss and significantly increased the number of multinucleated- or mega-nuclei by ~ 3.6-fold vs control. We tested five GTPase inhibitors and found that Y27632, a ROCK1/2 inhibitor, reverted the BPAF-induced MNG to the control level. Inhibition of Cdc42 by ML141 conversely increased the percentage of BPAF-induced MNG. mRNA expressions of genes p190RhoGap (Arhgap35) and MgcRacGap (Racgap1), important regulator of the GTPase activities during cleavage furrow formation, were also altered in the BPAF-treated C18-4 cells in a time-dependent manner. BPAF stimulated DNA synthesis in the C18-4 cells. In the cell cycle analysis, more than 50% of the BPAF-treated C18-4 cells became aneu- or poly-ploid (> 4N to > 8N), indicating a discordance in both cytokinesis and cell cycle checkpoint mechanisms. Pretreatment of Y27632 reduced the percentage of polyploidy cells. In addition, hierarchal cluster analysis of cytoskeletal proteins F-actins and ?-tubulins using machine learning-assisted HCA data confirmed that the treatment of Y27632 partially relieved the aberrated cytoskeletal structures and intensities induced by BPAF, suggesting a role of RhoA activity in the MNG formation. These results strongly implicated that BPAF may induce genomic instability in the male germ cells.
Abs#29. ACE2 turn-over in human airway epithelial cells
1Fan Huo, 1Lingxiang Zhu, 1Weifeng Liang, 1Ivana Kreso, 1Shelby Herrick, 2Dominik Schenten, 1Yin Chen
1. Department of Pharmacology and Toxicology, R. Ken Coit College of Pharmacy
2. Department of Immunology, College of Medicine, University of Arizona, Tucson, AZ 85721
Angiotensin-converting enzyme 2(ACE2), an important negative regulator of the renin-angiotensin-aldosterone system (RAAS), has been recently identified to be the receptor for SARS-CoV-2, the virus causing the current pandemic of coronavirus disease 2019 (COVID-19). However, the regulation of cellular level of ACE2 has not been completely understood. In this study, we found that the activation TACE could increase ACE2 shedding from the cell surface, and reciprocally, prolonged expression of ACE2 promoted pro-TACE cleavage (activation), further leading to enhanced ACE2 shedding. Furthermore, augmented ACE2 expression in human primary epithelial cells increased SARS-COV-2 infection and cellular interferon/proinflammatory signaling. Interestingly, SARS-COV-2 infection does- and time-dependently inhibited (until shut off) ACE2 at both protein and RNA levels. A complete understanding of mechanisms controlling cellular ACE2 turn-over will facilitate therapeutic development for treating COVID-19.
Abs#30. Immunological responses to Coccidioides infection
Ming Gu, Ziqi Yan, Lingxiang Zhu, Shelby Herrick, Ivana Kreso, Yin Chen
1. Department of Pharmacology and Toxicology, R. Ken Coit College of Pharmacy, University of Arizona, Tucson, AZ 85721
Coccidioides posadasii is a pathogenic fungus that is the causative agent of coccidioidomycosis, also known as valley fever in humans. Coccidioides have the characteristic of being thermally dimorphic, growing as mycelia in the soil and differentiating to spherule within mammals. The mature mycelia in the soil can form spores known as arthroconidia within the mycelium. Once a host inhaled spores and causes infection, arthroconidia transition into mature rupturing spherules within 4-6 days of infection. However, the pathological processes of coccidioidomycosis and the host immune response at the early stages of infection remain elusive. Based on our previous studies as well as other reports, type I interferon (IFN-I) plays a critical role in antifungal defense. Hence, in this study, we analyzed lung transcriptomic changes in C57BL/6 wile-type and IFNAR (type I interferon receptor 1) knockout mice at different time points (2 days, 5 days, and 10 days) post Coccidioides infection using RNA sequencing. Our results reveal the impact of Coccidioides on specific pathways and genes at different time points of infection. Pathways such as cilium movement, muscle contraction-related pathways, and airway mucins were upregulated, and pathway such as natural killer cell cytotoxicity and residential memory B cells were downregulated. Specifically, we found that, PLUNC (Palate, lung, and nasal epithelium clone protein), a lung protein that has been reported to have anti-bacterial and anti-inflammatory effects, were significantly upregulated in the early stage of Coccidioides infection via an IFN-I dependent manner. Furthermore, peptides from its active domain demonstrated potent antifungal activities. Overall, our data will add to the knowledge of host immunity against Coccidioides infection and reveal novel targets for the development of effective therapy to treat valley fever.
Abs#31. Sulforaphane (SFN) exhibits antiviral activity against SARS-COV-2
Weifeng Liang, Lingxiang Zhu, Ziqi Yan, Shelby Herrick, Ivana Kerso, Donna Zhang, Yin Chen
1. Department of Pharmacology and Toxicology, R. Ken Coit College of Pharmacy, University of Arizona, Tucson, AZ 85721
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still ongoing. Although vaccination rates are high nowadays, antiviral therapy that is readily available and easily administered to patients is still urgently needed to prevent severe disease, hospitalization, and death. So far, Molnupiravir and Paxlovid are the only two oral antiviral drugs authorized by the FDA for the treatment of COVID-19. SFN, a plant-derived chemical, particularly abundant in broccoli, cabbage, kale and brussels sprouts, has been well-documented on its antioxidant and anti-inflammatory effects. It also acts as a potent activator of NRF2. SFN has been reported to exhibit antiviral activity against SARS-CoV-2 in vitro and in vivo via an NRF2 independent mechanism. However, the underlying mechanism through which SFN inhibits SARS-COV-2 is not clear. In our study seeking to understand antiviral activities of NRF2 agonists, we found that SFN indeed inhibited SARS-COV-2 in Vero cells and in human lung epithelial cells. However, different from the other report, the lack of NRF2 reduced SARS-COV-2 replication and enhanced antiviral activity of SFN. Furthermore, using a fluorescence resonance energy transfer (FRET)-based assay, we found that SFN significantly inhibited the activity of non-structural protein 5 (NSP5), a main protease of SARS-COV-2. Therefore, anti-SARS-COV-2 activity of SFN is likely mediated by both NRF2-dependnent and independent mechanisms.
Abs#32. Transcriptional regulation of SCGB1A1 gene expression
Ziqi Yan, Weifeng Liang, Lingxiang Zhu, Shelby Herrick, Ivana Kreso, Yin Chen
1. Department of Pharmacology and Toxicology, R. Ken Coit College of Pharmacy, University of Arizona, Tucson, AZ 85718
Protein secretoglobin family 1A member 1(SCGB1A1) is a key component of homeostatic and reparative processes in the lungs. It is secreted as a pulmonary surfactant during lung tissue injury and can exert anti-inflammatory and anti-fibrotic functions. However, the transcription factors (TFs) that control the expression of the SCGB1A1 have not been systematically elucidated. We recently designed a new approach called ÒScreening of Transcriptional Operators and Regulators by Multi-omicsÓ (STORM). In STORM, we have queried multiple omics datasets including Chip-seq (by Cistrome Data Browser) and GEPIA. We discovered 42 TFs that might be potential transcriptional regulators of SCGB1A1 including FOXA1, FOXA2 and MYC among others. Further experimental confirmation of those TF candidates may provide useful insights into the pathogenesis and possible therapeutical targets of SCGB1A1-related lung illnesses including asthma, COPD or environmental arsenic exposure.
Abs#33. Impacts of polystyrene microplastics on the growth, survival, and macrophage differentiation of THP-1 cells
Kaitlin J. Martin1, Laura V. Santos-Medina1, Zachary A. Yee1, and Sebastian Medina1
1. Department of Biology, New Mexico Highlands University, Las Vegas, NM 87701Ê
Microplastics are environmental pollutants of increasing concern for human health. Humans are exposed to microplastics in a variety of ways including through drinking water. However, the immune and other health issues associated with microplastics exposures remain largely unknown. In the present study, the human monocytic cell line, THP-1, was exposed in vitro to increasing concentrations (0.1 µg/mL, 1 µg/mL, 10 µg/mL, and 100 µg/mL) of 1 µm, 5 µm, or mixed (1 µm + 5 µm) polystyrene microplastics, and the effects on viability, growth, and macrophage differentiation were evaluated. It was found that the growth of THP-1 cells was significantly reduced by exposures to high-doses of microplastics, independent of particle size. Interestingly, this suppression of THP-1 cell growth was not accompanied by significant alterations to cell viability with either the 1 µm, 5 µm, or mixed microplastics at any of the doses investigated. Additionally, the impacts of microplastics exposures on the macrophage differentiation of THP-1 cells was also evaluated. This study provides novel information regarding the immunotoxicity of microplastics, which is critical information for understanding the health impacts of these persistent and ever-increasing environmental pollutants.
Abs#34. Arsenic biotransformation is a key mediator of hematoxicity in vivo
Laura V. Santos-Medina1, Kaitlin J. Martin1, Zachary A. Yee1, Haikun Zhang2, Guanghua Wan2, Alicia M. Bolt2, Xixi Zhou2, Ke Jian Liu2, and Sebastian Medina1,2
1. Department of Biology, New Mexico Highlands University, Las Vegas, NM 87701
2. Department of Pharmaceutical Sciences, The University of New Mexico College of Pharmacy, Albuquerque, NM 87131
Human exposures to environmental metals such as arsenic are a worldwide public health concern. Chronic exposures to arsenic are linked to many health effects including anemia. Epidemiological studies in populations chronically exposed to arsenic have shown that methylation capacity, mediated by the arsenic (+III oxidation state) methyltransferase (As3MT) enzyme, is associated with elevated disease risk. Arsenic metabolism has an important role in detoxification, but it also generates bioactive intermediates with toxicity that may be greater than the parent inorganic arsenicals. Many studies suggest that the As3+ metabolite, monomethylarsonous acid (MMA3+) may be the major form of arsenic responsible for toxicity in vivo; however, few studies have been performed to directly evaluate this. In the present study, we used male As3MT knockout (KO) and wildtype C57BL/6J mice to evaluate the role of arsenic biotransformation in the development of anemia following drinking water exposures to As3+. We found that exposure to 1 mg/L (ppm) As3+ for 60 days resulted in the significant reduction of red blood cell counts, hemoglobin, and hematocrit in the blood of wildtype, but not As3MT KO mice. In support, we also observed significantly elevated levels of circulating erythropoietin in the serum of wildtype mice. Collectively, results from this study suggest that the process of arsenic biotransformation may have a critical role in mediating the hematotoxicity of arsenic.
Abs#35. Juvenile manganese exposure exacerbates neuroinflammation after viral infection
Casey P. McDermott1, Collin Bantle1, Savannah Rocha1,2, Ken Olson2, Ronald B. Tjalkens1
1. Toxicology Program, Dept. of Environmental and Radiological Health Sciences, Colorado State University , Fort Collins, CO
2. Dept. of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO
Parkinsons disease (PD) is a neurodegenerative movement disorder caused by the interaction of multiple factors, including age, environmental exposures, and genetic predisposition. The hallmarks of PD include inflammatory activation of glial cells, loss of dopaminergic neurons in the Substantia Nigra (SN), decreased striatal dopamine levels, and aggregation of alpha synuclein. Both manganese (Mn) exposure and infection with Western Equine Encephalitic Virus (WEEV) in animals and humans has previously been shown to cause neurological symptoms similar to PD. In this study, we examined how exposure to Mn during juvenile development could promote long-term neuroinflammation in astrocytes. To examine this phenomenon, we used a two-hit strategy with Mn and WEEV in transgenic mice with conditional knockout of the NF-?B inflammatory signaling pathway in astrocytes. We postulated that exposure to Mn in drinking water during juvenile development would accelerate neuroinflammation and neurodegeneration post-infection with WEEV. To test this hypothesis, mice were administered manganese chloride (MnCl2) in their drinking water from postnatal (PN) day 21 Ð PN51. Following this, mice were infected with WEEV intranasally at 3 months of age. Cellular markers of neuropathology were assessed in multiple brain regions by immunohistochemistry and immunofluorescence staining, including glial reactivity, protein aggregation, and neuronal loss. At 10 days post-infection, mice exposed to Mn as juvenile succumbed to neurotropic infection with WEEV, whereas astrocyte-specific NF-?B knockout mice all survived. There was a significant increase in microgliosis, astrogliosis, and dopaminergic neuron loss in the SN, entorhinal cortex and hippocampus that was ameliorated in KO mice. These data support the hypothesis that pre-treatment with manganese is a priming event for the innate immune system that intensifies the neuroinflammatory response to later infection with WEEV.
