bluuebunny/crossref_metadata_2025_split huggingface dataset:
Environmental Phthalate Diesters as hMT1 and hMT2 Melatonin Receptor Ligands
Authors: Jones Anthony J , Dubocovich Margarita L , Rajnarayanan Rajendram V 
Published: November 2023

Phthalates esters are commonly used plasticizers that have been linked to endocrine disruption and developmental alterations. Humans are exposed to these ubiquitous synthetic compounds via inhalation of ambient air, dermal contact with consumer products, and ingestion through food and drinking water. It is estimated from urinary metabolite levels that in the US, median adult daily exposure values for phthalate levels are about 8.5–16.8 μg/kg bw/day (Wittassek et al., Mol Nutr Food Res. 55:7–31, 2011). Using integrated pharmacoinformatics‐molecular docking approaches we discovered that several phthalate diesters show structural similarities to melatonin receptor ligands and bind with relatively high docking scores to melatonin binding pockets on in silico hMT1 and hMT2 melatonin receptor models. An assortment of phthalates including three monoesters and ten diesters with alkyl and/or aromatic substituents were tested for competition with 2‐[125I]‐iodomelatonin (100 nM) binding to hMT1 and hMT2 receptors stably expressed in CHO cells to assess binding affinity (Ki) as well as apparent intrinsic efficacy in the presence of GTP (100 μM). In GPCR binding assays, GTP decouples G proteins from receptors causing decreased affinity (rightward shift) for agonists but not for antagonists. Phthalate diesters with aromatic substituents (BBzP: Benzyl Butyl Phthalate; DPP: Diphenyl Phthalate) exhibited the highest affinities toward both hMT1 (BBzP Ki: 2.6 ± 0.3 μM, n=8; DPP Ki: 2.9 ± 0.7 μM, n=8) and hMT2 (BBzP Ki: 1.3 ± 0.2 μM, n=9; DPP Ki: 0.66 ± 0.10 μM, n=9) receptors. Among phthalates with alkyl substituents, four carbon (C4; DBP: Dibutyl Phthalate) and six carbon (C6; DHP: Dihexyl Phthalate) chain length diesters best competed for binding to hMT1 receptors with affinities ranging from 5 to 8 μM. hMT2 receptors did not show any preference to chain lengths of C6 or shorter (DEP: Diethyl Phthalate; DMP: Dimethyl Phthalate; DPrP: Dipropyl Phthalate; DBP; DHP), as all Ki values were between 2 and 7 μM. DBP and BBzP bound to melatonin receptors with affinities in the low micromolar range, however their respective monoester metabolites, MBP (Monobutyl Phthalate) and MBzP (Monobenzyl Phthalate), did not compete for binding to either receptor. The presence of GTP caused a leftward shift of the binding competition curves for both BBzP and DPP at hMT1 melatonin receptors (BBzP KiGTP/KiControl: 3.8; DPP KiGTP/KiControl: 2.6) suggesting inverse agonist efficacy. Little or no shift (BBzP KiGTP/KiControl: 1.3; DPP KiGTP/KiControl: 1.5) on competition curves for hMT2 melatonin receptors suggests antagonist efficacy. Together these data suggest that phthalate diesters could interact with hMT1 and hMT2 receptors to disrupt melatonin receptor‐mediated signaling processes in target tissues.Support or Funding InformationSupported by ES 023684 to MLD and RVR.
Acetate, an Ethanol Metabolite Increases Neuroinflammation and Neuronal Death: Implications in Ethanol Neurodegeneration
Authors: Chapp Andrew D. , Driscoll Kyle M. , Behnke Jessica , Shan Zhiying , Chen Qinghui 
Published: November 2023

Upregulation of neuroinflammatory signaling molecules following ethanol consumption has been identified as a key cellular mechanism to contribute to neuronal excitotoxicity. Evidence indicates that ethanol excitotoxicity occurs during ethanol withdrawal/clearance, times at which acetate concentrations are still elevated. Studies have demonstrated that acetate is a very active and powerful metabolite of ethanol, which may be one of the key components to underlie the mechanisms of neuronal excitotoxicity/apoptosis induced by ethanol consumption. Here, we tested the hypothesis that acetate increases neuroinflammatory cytokines, which may contribute to neuronal excitotoxicity/apoptosis and enhanced cellular death. Whole brain neuronal cultures incubated with acetate (2 mM) for 3 hrs. increased mRNA levels significantly (p < 0.05 vs control) of tumor necrosis factor alpha (TNFα), interleukin‐1β (IL‐1β) and fos related antigen 1 (fosl1); 2 fold, 8 fold and 2 fold respectively. Furthermore, 24 hr. treatment with acetate (2 mM) in whole brain neuronal cultures followed by staining with propidium iodide and analyzed via flow cytometry (1000 neurons, triplicates) showed significant neuronal death (p < 0.05 vs control), acetate (62.3 %) vs control (25.5 %). In a separate treatment, whole brain neuronal cultures were incubated with acetate (2 mM) for 24 hrs. and then stained with Fluo‐4AM (calcium dye) and analyzed via flow cytometry (1000 neurons, triplicates) and showed a significant 5 % increases in cytosolic calcium (p < 0.05 vs control). This data suggests that physiological circulating levels of acetate (2 mM) following ethanol consumption and metabolism lead to increased neuroinflammation which enhances whole brain cellular apoptosis/death and may be the underlying cause of alcohol associated neurodegeneration.Support or Funding InformationAHA 16PRE27780121, Andrew Chapp, AHA 11SDG7420029, Zhiying Shan; R15‐HL122952, Qinghui Chen
Factors Impacting the Estimation of Renal Clearance of N1‐methylnicotinamide, an Endogenous Probe for Renal Cation Transporters
Authors: Bergagnini‐Kolev Mackenzie C. , Hebert Mary F. , Easterling Thomas R. , Lin Yvonne S. 
Published: November 2023

N1‐methylnicotinamide (1‐NMN) has been previously investigated as an endogenous probe for the activity of renal transporter organic cation transporters 2 (OCT2) and multidrug and toxin extrusion proteins 1 and 2‐K (MATE1 and MATE2‐K). In drug‐drug interaction studies, 1‐NMN renal clearance was shown to decrease after the administration of OCT2, MATE1, and MATE2‐K inhibitors. We have shown that pregnancy increases the renal clearance of 1‐NMN. We investigated the factors that may impact the estimation of renal clearance using 1‐NMN such as OCT2 genotype, as well as urine and plasma collection interval duration (8, 12 and 24 hours). In addition, we assessed whether 1‐NMN renal clearance as determined by spot urine/plasma measurements or longer collection intervals were comparable under basal and induced states. Secondary analysis was conducted on blood and urine samples collected from women prescribed metformin during early‐, mid‐, and late‐pregnancy and postpartum and healthy volunteers administered rifampin. Samples were analyzed for 1‐NMN using liquid chromatography‐mass spectrometry. The renal clearance of 1‐NMN was estimated and correlated with metformin renal clearance when data were available. 1‐NMN renal clearance was higher in both mid‐ (504 ± 293 mL/min, p < 0.01) and late‐pregnancy (557 ± 305 mL/min, p < 0.01) compared to postpartum (240 ± 106 ml/min). Metformin renal clearance and 1‐NMN renal clearance were positively correlated (rs = 0.68, p < 0.0001). OCT2 G808T genotype did not appear to affect 1‐NMN renal clearance. Plasma 1‐NMN levels were highly variable throughout the day, thus a single plasma measurement was not sufficiently representative of Css,1‐NMN to estimate 1‐NMN clearance. Studies looking at the impact of urine and plasma collection interval duration and spot sampling are ongoing. As an endogenous biomarker, 1‐NMN may be useful in assessing changes in renal OCT2, MATE1, and MATE2‐K activity, however sample collection and other subject‐specific factors (e.g., diet) may impact the results.Support or Funding InformationThis work was supported in part by the Eunice Kennedy Shriver National Institute of Child Health & Human Development [Grant U10HD047892]; NIH National Center for Advancing Translational Sciences through the Clinical and Translational Science Awards Program (CTSA) [Grant UL1TR000423]; and the NIH National Institute of General Medical Sciences [Grant T32GM 007750]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Eunice Kennedy Shriver National Institute of Child Health & Human Development or the National Institutes of Health.
Prioritizing Pharmacokinetic Drug Interaction Precipitants in Natural Products: Application to OATP Inhibitors in Grapefruit Juice
Authors: Johnson Emily J , Won Christina S , Köeck Kathleen , Paine Mary F 
Published: November 2023

Background and objectiveHerbal and other natural products typically contain multiple constituents that can precipitate adverse pharmacokinetic interactions with conventional drugs. Streamlined approaches are needed to prioritize candidate natural product precipitants during early investigation of these complex interactions. Grapefruit juice is a well‐characterized natural product that increases systemic exposure to several drugs via inhibition of organic anion‐transporting polypeptide (OATP) uptake in the intestine. Using grapefruit juice as a model natural product, a streamlined approach was developed to prioritize OATP inhibitors as precipitants of pharmacokinetic grapefruit juice‐drug interactions.MethodsOATP2B1‐expressing MDCKII cells and the probe substrate estrone 3‐sulfate were used to assess the inhibitory effects of candidate constituents representative of the flavanone (naringin, naringenin, hesperidin), furanocoumarin (bergamottin, 6′,7‐dihydroxybergamottin), and polymethoxyflavone (nobiletin, tangeretin) classes contained in grapefruit juice. IC50s were recovered via nonlinear least‐squares regression analysis. Intestinal absorption simulations based on physiochemical properties were used to estimate the unbound intracellular enterocyte concentration (Ient) of each constituent. Two ratios were determined for each constituent: Ient/IC50 and maximum reported concentration in grapefruit juice to IC50 (IGFJ/IC50). Constituents were ranked in order of drug interaction liability according to pre‐defined cutoff values (Ient/IC50, 0.1; IGFJ/IC50, 10).ResultsNobiletin was the most potent OATP2B1 inhibitor (IC50, 3.7 μM); 6′,7‐dihydroxybergamottin, naringin, naringenin, and tangeretin were moderately potent (IC50, 22–39 μM); and hesperidin and bergamottin were the least potent (IC50 >300 μM). The Ient/IC50 for naringin, naringenin, nobiletin, and tangeretin exceeded 0.1. The IGFJ/IC50 for naringenin and naringin exceeded 10.ConclusionsBoth Ient/IC50 and IGFJ/IC50 correctly prioritized the flavanones, and excluded the furanocoumarins, as precipitants of intestinal OATP‐mediated grapefruit juice‐drug interactions in humans. The more sensitive Ient/IC50 identified two polymethoxyflavones as potential additional inhibitors. A similar approach could be applied to other natural products for initial prioritization of constituents as potential precipitants of pharmacokinetic natural product‐drug interactions.Support or Funding InformationThis work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant R01 GM077482] and National Center for Advancing Translational Sciences [Grant UL1TR001111]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Analysis of Liver Toxicity Markers in Danio rerio following Ethanol Exposure
Authors: Chiari John B , Charles Jamie M , Laperche Jacob M , McGinnis Courtney L 
Published: November 2023

Zebrafish have been used for many years in developmental and reproductive biology, yet little is still known about the adult zebrafish liver. Additionally, many of the mammalian liver cell types have yet to be characterized in zebrafish, including Kupffer cells, oval cells, and stellate cells. Also, there is minimal information available regarding zebrafish liver transporters and nuclear receptors. Therefore, it is important to understand the mechanism of xenobiotic metabolism in zebrafish to support their use as a model organism for studying the fate and biological effects of xenobiotics. The value of zebrafish as a model organism is based on a broad set of gene similarities (among fish species and humans) involved in metabolism and transportation of endogenous and exogenous chemicals. While there are molecular similarities between zebrafish and humans, there are also differences that may impact how compounds are metabolized. Therefore, it is absolutely necessary to investigate and validate the assumptions that zebrafish possess conserved genes, enzymes, and processes involved in all stages of metabolism. This study investigates the impact of ethanol exposure on liver toxicity markers in adult Danio rerio. Liver tissue from sham and ethanol injected fish were collected, pooled and RNA was isolated for qPCR. Relative expression levels for each gene were determined and normalized to βactin. Of the seventeen genes that were assessed, our results demonstrate a significant upregulation of the following genes following ethanol treatment: abcb4 (mdr3), abcc1(mrp1), abcc4(mrp4), abcc9(sur2), abcg5, mate1, oatp1 and slc4a2(ae2). Alcohol metabolism increases acetate production, a precursor for cholesterol and fatty acid synthesis. Historically, this increase in acetate levels driven by alcohol, is a contributor of hepatic steatosis (fatty liver). In a previous study conducted in the lab, zebrafish chronically exposured to ethanol displayed hallmark signs of hepatic steatosis. An upregulation in slc4a2, a sterol and cholesterol transporter aimed to eliminate dietary cholesterol, was found, consistent with the digestion of high levels of acetate. Furthermore, upregulation of abcc4, a prostaglandin transporter, is indicative of oxidative stress within the liver, caused by ethanol metabolism. Additionally, the upregulation of the other liver transporters would be consistent with normal hepatic ion flow during xenobiotic methabolism.Support or Funding InformationQuinnipiac University: College of Arts and Sciences
In Silico Molecular Dynamics Analysis of Dopamine Transporter Conformational Preference as a Function of Occupancy with Inhibitors of Low Abuse Potential
Authors: Wasko Michael J. , Madura Jeffry D. , Hong Weimin C. , Katz Jonathan L. , Surratt Christopher K. 
Published: November 2023

The plasmalemmal dopamine transporter (DAT) protein removes dopamine from the extracellular space via electrogenically‐driven uptake, and is a primary target of the abused psychostimulants cocaine and the amphetamines. Certain “atypical” high‐affinity DAT inhibitors such as benztropine and GBR12909 do not share cocaine's abuse potential, and it has been postulated that a preference for the inward‐ (cytoplasmic‐) facing DAT conformation during the substrate translocation cycle correlates with low abuse potential. The objective of this study was to characterize for in vitro and in silico conformational preference several N‐substituted benztropine analogs (GA169, GA250, GA299, JHW013) that have been characterized as atypical because they bind the DAT inhibit dopamine uptake but have behavioral effects that differ from those of cocaine. A cysteine accessibility assay probed each drug's preference for the outward‐facing conformation of DAT. Computational models of the human DAT in outward‐ or inward‐facing conformations were created in MOE using the Drosophila DAT crystal structure 4M48 template. DAT inhibitors were docked to the optimized DAT models, followed by 400 ns molecular dynamics (MD) simulations using ACEMD software to ascertain a conformational preference for each ligand. Whereas cocaine showed its characteristic preference for the outward‐facing conformation in the cysteine accessibility assay, benztropine as well as the (R)‐2″‐amino‐3″‐methyl‐n‐butyl (GA250), indole‐3″‐ethyl (GA169), and cyclopropylmethyl (JHW013) analogs showed preference for the inward‐facing conformation, in keeping with their atypical effects. In contrast, the 2″‐aminoethyl analog (GA299) showed no conformational preference despite its atypical effects. Analysis of the MD simulations, using the external and internal gate residues as a reference, confirmed that cocaine and benztropine achieved their preferred conformation. The initial outward‐facing DAT simulation formed an occluded structure with benztropine in the S1 pocket, while the external gate remained open during the cocaine simulation. The outward‐facing simulation with GA299 shared characteristics with cocaine, while GA169 displayed similarities with benztropine bound to the inward‐facing structure. In summary, enough exceptions were observed to question the proposed correlation between a DAT inhibitor's abuse potential and its preference for the inward‐ or outward‐facing DAT conformation. This work suggests that in silico studies on DAT protein‐inhibitor complexes provide a new tool for analysis of physiologically‐relevant drug receptor conformations and for guiding therapeutic drug design.Support or Funding InformationFunding: ZIA DA000103‐26 (IRP, NIDA), DA027806, XSEDE MCB060069, Butler Faculty Startup Fund, Holcomb Research Award.
Role of breast cancer resistance protein in obesity associated chronic low grade inflammation
Authors: Mishra Jayshree , Kumar Narendra 
Published: November 2023

ObjectiveThe major immunological manifestation in diabetes/obesity is characterized by chronic low grade inflammation that leads to altered drug metabolism and disposition. Alteration in drug metabolism has profound impact on the pharmacotherapy of widely used clinically relevant medications in terms of safety and efficacy and increased toxicity. Altered drug metabolism and disposition is associated with the alteration in the expression and activity of the several key drug metabolizing enzymes (DMEs) and transporters. However, the role of inflammation in regulation of drug transporters in diabetes/obesity remains unclear. Breast cancer resistance protein (BCRP) is a principal drug transporter protein expressed in intestine, liver and brain extrudes a broad range of xenochemicals and to protect the organism against xenotoxicity by facilitating excretion, and limiting absorption of potentially toxic substrate molecules, including many chemotherapeutic drugs, the possible involvement of BCRP in obesity is poorly understood. It is critical to understand the mechanisms that regulate mucosal detoxification mediated by BCRP under normal physiological conditions and under the chronic low grade inflammation as seen in obesity. A better understanding of the mechanistic details underlying the regulation of BCRP in obesity will contribute towards development of powerful in vitro and in vivo tools in predicting the drug response and opt for better drug design and development.Materials and MethodsFreshly dissected human colon tissue from normal and obese were immunostained and analyzed for BCRP (Novus). Phosphorylation status of this protein in both normal and obese were assessed through immunoprecipitation. Functional activity of BCRP was measured using Efflux id Gold kits from Enzo Life sciences.Summary of ResultsOur results show that colonic epithelial cells of normal subject express BCRP, however there is a substantial decrease in expression of BCRP in obese patients. Also in obese patients there is a substantial decreases in the tyrosine phosphorylation status of BCRP. Expression and activity of BCRP was found to be significantly reduced in the induced with high fat diet.ConclusionOur data suggest that colonic BCRP plays an important role in obesity. Further, our goal is to facilitate a better understanding of the mechanistic role of BCRP in the process of chronic low grade inflammation and associated chronic diseases.Support or Funding InformationHealth‐ SBIR (GM109528)
Polarization of Adipose‐Derived Mesenchymal Stem Cells into Immunomodulating Phenotypes
Authors: Rivera‐Cruz Cosette M. , Shearer Joseph J , Figueiredo Marxa Leao 
Published: November 2023

Mesenchymal stem cells (MSCs) have been studied often to assess their potential use in cell therapy because of their capacity to differentiate into non‐hematopoietic cell lineages, modulate immune responses, and promote tissue repair and regeneration. In addition, MSCs have been found to have roles in the promotion or repression of tumor growth and tumor cell migration. However, the mechanisms by which MSC affect tumor cells are not fully understood. Studies with bone marrow‐derived MSCs have shown that these cells can be polarized by Toll‐like receptor (TLR) signaling into two phenotypes with opposite effects on tumor development1. TLR4 signaling induces an immune activating MSC1 population, whereas TLR3 signaling induces an immune suppressive MSC2 population2. Adipose‐derived mesenchymal stem cells (ASCs) are important constituents of tumor microenvironments. Their ability to migrate into tumors make these cells a potential candidate for delivering immunotherapy against tumor progression. Although the MSC1 and MSC2 duality has not been reported for ASCs, we hypothesized that due to their similarity to bone marrow MSCs, that ASC may share the polarization phenotype. In order to test this hypothesis, we conducted preliminary experiments using ASC priming with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (Poly (I:C)) and assessed gene expression changes that would be consistent with polarization using real time PCR. These experiments have shown expression of target TLRs in human ASCs from two independent donors. Further studies are in progress, including priming with TLR agonists, gene expression analysis to confirm priming (IL‐6 for ASC1; CCL5 for ASC2), and assays to assess whether ASC1 or ASC2 conditioned media promote or inhibit prostate tumor cell colony formation.Support or Funding InformationWe acknowledge support from the Purdue PREP program (DTA Transformative Award) and statrt up funds from the BMS department
Inhibition of Neuropilin 1 signaling in glioma associated macrophages and microglia slows tumor progression
Authors: Miyauchi Jeremy T , Chen Danling , Choi Matthew , Shroyer Kenneth , Selwood David , Tsirka Stella 
Published: November 2023

Gliomas are the most commonly diagnosed primary tumors of the central nervous system (CNS). Median times of survival are dismal regardless of the treatment approach, underlying the need to develop more effective therapies. Modulation of the immune system is a promising strategy as innate and adaptive immunity play important roles in cancer progression. Glioma associated microglia and macrophages (GAMs) can comprise over 30% of the cells in glioma biopsies. Gliomas secrete cytokines that suppress the anti‐tumorigenic properties of GAMs, causing them to secrete factors that support the tumor's spread and growth. Neuropilin 1 (Nrp1) is a transmembrane receptor that in mice both amplifies pro‐angiogenic signaling in the tumor microenvironment and affects behavior of innate immune cells. Using a Cre‐lox system, we generated mice that lack expression of Nrp1 in GAMs. We demonstrate, using an in vivo orthotopic glioma model, that tumors in mice with Nrp1‐deficient GAMs exhibit less vascularity, grow at a slower pace, and are populated by increased numbers of anti‐tumorigenic GAMs. Moreover, glioma survival times in mice with Nrp1‐deficient GAMs were significantly longer. Treating wild‐type mice with a small molecule inhibitor of Nrp1's b1 domain, EG00229, which we show here is selective for Nrp1 over Nrp2, yielded an identical outcome. Moreover, transplanting bone marrow from mice with Nrp1‐deficient macrophages to wild type recipient mice led to reductions in tumor growth, al‐be‐it not to the extent seen in knockout mice. Nrp1‐deficient or EG00229‐treated wild‐type microglia exhibited a shift towards anti‐tumorigenicity as evident by altered inflammatory marker profiles in vivo and decreased SMAD2/3 activation when conditioned in the presence of glioma‐derived factors. These results provide support for the proposal that pharmacological inhibition of Nrp1 constitutes a potential strategy for suppressing glioma progression.Support or Funding InformationThis work was funded by the following award from the NIH: NCI F30CA196110