bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Genotype, Age and Gender on Hepatic Abundance of Flavin Monooxigenase 3
Authors: Xu Meijuan , Bhatt Deepak Kumar , Chaw Katrina , Yeung Catherine , Prasad Bhagwat 
Published: November 2023

Background and PurposeHepatic flavin monooxigenase 3 (FMO3) metabolizes a broad array of nucleophilic heteroatom‐containing drugs (e.g., amphetamine, sulindac, benzydamine, ranitidine, tamoxifen and ethioniamide), endogenous and exogenous chemicals (e.g., catecholamine, trimethylamine and nicotine) and pesticides. To predict the effect of genotype, age and gender on the hepatic metabolism of FMO3 substrates, we quantified FMO3 protein abundance in human liver microsomes by LC‐MS/MS proteomics.MethodsHuman liver microsomes were isolated from the tissue samples procured from University of Washington (UW, n=39) and St. Jude Children's Hospital (SJLB, n=249). Two peptides, i.e., VAIIGAGVSGLASIR (quantifier) and NNLPTAISDWLYVK (qualifier) were selected for LC‐MS/MS quantification. Total protein concentration was determined and the samples were denatured, reduced, alkylated, desalted and digested as previously reported (1, 2). FMO3 was quantified by AB Sciex 6500 triple quadrupole MS/MS using optimum conditions of reproducibility (1). Genotyping and mRNA expression analyses were performed using previously reported methods (3). The functional activity of FMO3 in selected human liver microsomes (UW samples) was quantified by using benzydamine as a probe substrate.ResultsGenotype and age significantly affect FMO3 protein abundance and activity. Out of the common twenty‐one FMO3 SNPs, six SNPs (rs2064074, rs28363536, rs2266782, rs909530, rs2266780 and rs909531) were associated with significantly decreased protein abundance. However, there was no correlation of these SNPs on the mRNA expression of FMO3 perhaps due to the effect of these SNPs on post‐translational modification, e.g., proteosomal degradation. Based on the activity data on representative samples, human livers harboring variant allele of rs2266780 showed no in vitro metabolic activity toward benzydamine, indicating effect of this SNP on both substrate affinity (Km) and FMO3 protein abundance. Consistent with our previous data (4), infants and children have significantly lower FMO3 abundance as compared to adults. FMO3 abundance was not associated with gender.ConclusionsFMO3 protein abundance is highly affected by genotype and age but is independent of gender. These quantitative data are important in predicting FMO3 mediated hepatic N‐oxidation or S‐oxidation of endogenous biomolecules and xenobiotics.Support or Funding InformationNICHD R01HD081299‐02
Nicotine metabolism by the flavin monooxygenase (FMO) family of enzymes.
Authors: Perez‐Paramo Yadira , Watson Christy , Chen Gang , Lazarus Philip 
Published: November 2023

Nicotine is the addictive tobacco‐specific alkaloid present in cigarettes. Nicotine is metabolized in vivo primarily by hydroxylation by CYP2A6, but is also glucuronidated by UDP‐glucuronosyltransferases (UGTs) and oxidized by FMOs. Nicotine‐N‐oxide has been detected in smoker's urine, accounting for 4–8% of total urinary nicotine metabolites. While previous studies have suggested that the hepatic FMO3 is the principal enzyme involved in nicotine‐N‐oxidation, a comprehensive analysis of nicotine oxidation by all FMOs has not previously been performed. To characterize the catalytic activity of FMOs in the formation of nicotine‐N‐oxide, wild‐type FMOs 1 through 5 were individually overexpressed in HEK293 cells and used in oxidation reactions containing between 0.1 – 5000 μM of (−)‐nicotine and NADPH‐regenerative system; nicotine‐N‐oxide formation was monitored by UPLC‐MS/MS. While all five FMOs exhibited oxidation activity against nicotine, only FMO1 exhibited a KM of less than 1 mM (0.76 mM), with FMOs 3, 4 and 5 all exhibiting a KM of >40 mM. While FMO2 is expressed primarily in the lung and possibly brain, this enzyme is functional only in people of African ancestry due to a SNP in the FMO2 stop codon (codon 472; MAF=0.15); most of the remaining human population expresses a truncated non‐functional enzyme. Functional FMO2427Gln also exhibited low activity against nicotine, with a KM = 71 mM. Non‐synonymous polymorphic variants for FMO1 and FMO3 have been overexpressed in HEK293 cells and are currently being screened for activity against nicotine in vitro. These data are consistent with FMO1 being the principal enzyme involved in nicotine‐N‐oxidation and with previous studies demonstrating that functional genetic variants in FMO1 are associated with differences in nicotine dependence.Support or Funding Information

National Institutes of Health, National Institutes of Environmental Health Sciences [Grant R01‐ES025460]
Health Sciences and Services Authority of Spokane, WA [Grant WSU002292]
ecoAO: A Simple System for the Study of Drug Metabolism by Human Aldehyde Oxidase
Authors: Paragas Erickson Mesina , Humphreys Sara , Min Joshua , Joswig‐Jones Carolyn , Jones Jeffrey 
Published: November 2023

Aldehyde oxidase (AO) is a cytosolic enzyme responsible for the metabolism of some drugs and drug candidates. AO catalyzes the oxidative hydroxylation of substrates including several aliphatic and aromatic aldehydes, and nitrogen‐containing heterocyclic compounds. Although AO plays a significant role in human Phase I drug metabolism, its role in drug clearance is not well understood. Major reasons for its recalcitrance to examination are two‐fold. First, established in vitro systems used to study AO, including liver cytosol, pure enzyme, S9 fractions, and primary and cultured hepatocytes, display hyper‐variability in their AO expression and activity profiles. Second, AO metabolite standards are often difficult to chemically synthesize due to the chemo‐ and region‐selectivity of the enzyme. In this study, we describe a simple, economical, Escherichia coli system – ecoAO – that addresses both issues simultaneously. ecoAO contains pTHco‐hAOX1 (Silke Leimkühler, University of Potsdam), a codon‐optimized and lactose‐inducible human aldehyde oxidase‐containing plasmid, expressed in Moco‐producing E. coli strain TP1017 (Tracy Palmer, University of Dundee). Our direct cell paste assays exhibit specific AO activity with a range of known substrates (DACA, zaleplon, zoniporide and O6‐benzylguanine) and candidate substrates (quinoline series) against a non‐mammalian background. We also demonstrate an important application of the ecoAO system, the biotransformation of AO substrates, yielding significant quantities of metabolite (~30% yield) upon purification.Support or Funding InformationThis work was supported by the National Institutes of Health National Institute of General Medical Sciences grant GM100874.
Comparison of Cytochrome P450 3A4 and 3A7 with Azole Inhibitors
Authors: Godamudunage Malika P. , Lampe Jed N. , Scott Emily E. 
Published: November 2023

Cytochrome P450 (CYP) 3A enzymes metabolize up to 50% of human drugs. While CYP3A4 is the major adult enzyme, CYP3A7 is the major form through 6–12 months of development. Though 87% identical and frequently assumed to be functionally very similar, it has recently become clear that there are some significant differences between CYP3A4 and CYP3A7 that may affect fetal and infant drug metabolism. This knowledge gap motivates a direct study of CYP3A7 metabolism and inhibition to better inform drug usage in very young patients.Azoles can be effective for common fungal infections in infants, but can also be rather nonselective CYP inhibitors. In addition to the clinical relevance of these compounds, azoles constitute a useful series of CYP3A active site probes. This study systematically evaluated the interactions of a series of azoles with purified, recombinant human CYP3A4 and CYP3A7. Binding studies revealed that each of the following azoles demonstrated spectral features typical for direct coordination to the heme iron (type II binding) with parallel rank order affinities for the two enzymes: from subnanomolar Kd values for clotrimazole, to moderate affinity for liarozole and ketoconazole, and low affinity for imidazole. In contrast, fluconazole and fosfluconazole demonstrated more unusual binding characteristics that differed between the two CYP3A enzymes. Fluconazole binding to CYP3A7 resulted in only increasing absorbance, migrating at higher concentrations to wavelengths associated with heme coordination. In contrast, fluconazole binding to CYP3A4 was characteristic of water displacement from the heme (type I) at low concentrations, but converted to heme coordination at high concentrations, suggesting that CYP3A4 may bind fluconazole in different orientations, perhaps depending on whether one fluconazole molecule or two are bound in the active site simultaneously. This would be consistent with the established capacity of CYP3A4 to bind more than one azole molecule simultaneously. Fosfluconazole, a more water‐soluble phosphorylated version of fluconazole, demonstrated a red‐shifted type I spectrum with CYP3A4, but with CYP3A7 yielded only increasing absorbance consistent with water displacement from the iron. Overall, these observations establish differences between CYP3A7 and CYP3A4 interactions with fluconazole and fosfluconazole, prompting further evaluation by comparative inhibition studies and structural studies. Such differences may underlie differential metabolism of common drugs at different life stages and inform dosing in infants vs. adults.Support or Funding InformationNational Institutes of Health R37GM076343 (EES)
Benzop(a)pyrene in Cigarette Smoke Enhances HIV‐1 Replication in Monocytic Cells: Potential Role of Cytochrome P450s and Oxidative Stress
Authors: Ranjit Sabina , Midde Narasimha M. , Sinha Namita , Kumar Santosh 
Published: November 2023

Smoking aggravates HIV‐1 pathogenesis and leads to decreased responses to antiretroviral therapy. However, the exact mechanism by which smoking enhances HIV‐1 pathogenesis is unknown. Benzo(a)pyrene (BaP) is a major carcinogen in cigarette smoke. It requires metabolic activation through cytochrome P450s (CYPs) to exert its toxic effects. We hypothesize that BaP is metabolized by CYP‐mediated pathway and produce reactive oxygen species (ROS). Oxidative stress resulting from increased ROS would aggravate HIV‐1 replication. Recently, we have shown that chronic exposure of BaP to U937 monocytic cells results in the significant induction of CYPs (CYP 1A1 and 3A4) and antioxidants (catalase and SOD1), and increase in the generation of ROS and cellular toxicity. We also confirmed these results in human primary macrophages. Next, we examined the chronic effect of BaP in U1 cells (HIV‐1 infected U937 monocytic cells). When U1 cells were exposed to BaP for seven days, we observed ~4 fold increase in HIV‐1 replication. In addition, there was a significant increase in the expression of CYP1A1 at mRNA level as well as there was an increase in CYP1A1 enzymatic activity. Elevated ROS and massive cell death were also observed. In order to confirm the role of CYP1A1 and 1B1 in BaP‐induced oxidative stress pathway and HIV‐1 replication, we knocked down the gene expression of CYP1A1 and 1B1 in HIV‐1 infected human primary macrophages, using SiRNA. Our results showed decrease in the viral replication in CYP1A1 and 1B1 knock‐out cells upon BaP exposure. We will determine oxidative DNA damage in untreated, and CYP1A1 and 1B1 knock‐out cells using TUNNEL assay. We are now in the process of determining the role of oxidative stress mediated through CYP pathway in HIV‐1 replication. For this, we will expose HIV‐infected primary macrophages with general and specific antioxidants, followed by exposure to BaP. Finally, we will quantify BaP metabolites in U1 cells and/or HIV‐1 infected human primary macrophages using Liquid Chromatography‐Mass Spectrometry. The results from the present work are clinically relevant as they would help to find a novel therapeutic target to improve drug therapy outcomes in HIV positive smokers.Support or Funding InformationDepartment of Pharmaceutical Sciences, University of Tennessee Health Science Center
Evaluation of Seahorse XF Cell Mito Stress Test for Detection of Mitochondrial Dysfunction Using Prototypical Mitochondrial Toxicants in the PC‐3 Cell Line
Authors: Zhang Jie L , Krause Caitlin , Ciurlionis Rita , Sharapova Tatiana L , Wilsbacher Julie , Ackler Scott , Van Vleet Terry , Liguori Michael J 
Published: November 2023

Mitochondrial dysfunction has been frequently implicated in some drug‐induced toxicities. Various methods for the detection of mitochondrial toxicity are available such as an oxygen consumption assay to measure mitochondrial basal respiration, assays to measure direct effects of drugs on the five complexes in the mitochondrial electron transport chain (ETC), and assays to detect changes in mitochondrial DNA (mtDNA) copy number or changes in mitochondrial membrane potential. The Seahorse technology platform has emerged as a new technique in assessing mitochondrial respiration in cells, where basal, ATP‐linked, maximal and non‐mitochondrial respiration can be measured in the same experiment. Spare respiratory capacity (SRC) is calculated as the difference between maximal and basal respiration. In this study, Seahorse XF Cell Mito Stress Test was evaluated in the PC‐3 cell line using tool compounds known to directly inhibit the complex or complexes in the ETC. Cells were acutely treated with amiodarone (5–80 μM), simvastatin (5–75 μM), tamoxifen (0.625–20 μM), troglitazone (2.5–50 μM), penicillin (12.5–100 μM, negative control), and vehicle (0.1% DMSO). Exposure of these tool compounds revealed an immediate and pronounced decrease in SRC with a ranking of (greatest to lowest inhibition) troglitazone (IC50< 2.5 μM)> amiodarone (IC50<5 μM) >tamoxifen (IC50 = 17.7 μM) >simvastatin (IC50 = 20.7μM).Tool compounds known to cause mitochondrial toxicity through an indirect mechanism were also explored in the Seahorse platform. 2′, 3′‐dideoxycytidine (ddC), an inhibitor of mtDNA synthesis, and chloramphenicol (CAM), an inhibitor of mitochondrial protein synthesis, were tested. A 3‐day pre‐treatment of ddC (0.12–30 μM) and CAM (0.04–10 μM) showed a decrease of SRC in PC‐3 cells with IC50=0.39 μM and 2.7 μM respectively. The decrease of SRC was further supported by an in‐cell ELISA assay where a set of proteins in the ETC complexes was measured. A decrease of mitochondrial encoded COX‐1 to nuclear encoded SDH‐A protein ratio was observed after a 5‐day treatment of ddC and CAM in PC‐3 cells with IC50=0.016 μM and <0.1 μM respectively. In the dose ranges tested, the 3 or 5 day pre‐treatment of ddC and CAM showed minimal cytotoxicity in the PC‐3 cells. In conclusion, the Seahorse XF Cell Mito Stress Test is a sensitive assay to evaluate mitochondrial dysfunction using a set of tool compounds, and this new method combined with traditional methods will be helpful to better understand the mechanism of mitochondrial toxicity in the development of new drugs.Support or Funding InformationThe design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
GPCRs as novel potential therapeutic targets in cancer
Authors: Insel Paul A , Sriram Krishna , Wiley Shu Zhou , French Randall P , Lowy Andrew M 
Published: November 2023

G protein‐coupled receptors (GPCRs) are the most widely targeted class of FDA‐approved therapeutics but in cancer, GPCRs are generally only targeted in endocrine tumors. We hypothesize that GPCRs have much broader importance in cancer and that therapeutics directed at GPCRs may have unappreciated utility in the treatment of a wide variety of cancers. To test this hypothesis, we have used unbiased approaches (Taqman GPCR arrays and RNA‐seq), mining of publicly available datasets (such as The Cancer Genome Atlas, TCGA), and validation studies to assess GPCRs in multiple human tumors, cancer cells and stromal cells (cancer‐associated fibroblasts, CAFs). A major focus has been on pancreatic ductal adenocarcinoma (PDAC) tumors and cells and pancreatic CAFs (PCAFs). We find that PDAC cells and PCAFs express >75 different GPCRs, including many orphan GPCRs (without known physiologic agonists) and that a substantial number of these GPCRs are expressed at much higher levels than in normal precursor cells. A cluster of GPCRs is overexpressed in PDAC tumors. Two of these receptors are orphan GPCRs (Orphan 1 and Orphan A), neither of which is mutated but each is highly expressed in PCAFs and PDAC cells, respectively. Both Orphan 1 and Orphan A have at least 2‐fold higher expression in >90% of PDAC tumors in TCGA compared to that of normal pancreas. Higher expression of orphan A is associated with increased mortality and a worse clinical course and Orphan 1 expression correlates with that of multiple fibrotic genes. Importantly, both Orphan 1 and Orphan A are functional and appear to contribute to the malignant phenotype. For example, siRNA knockdown of Orphan 1 in PCAFs blunts production of profibrotic markers and decreases ability of conditioned media from PCAFs to enhance proliferation of PDAC cells. Together, these and other results suggest that GPCRs represent previously unrecognized contributors to cancers besides endocrine tumors and as such, may be druggable, novel targets for the treatment of cancer, including pancreatic cancer, a tumor for which new therapeutics are a critical, unmet need.Support or Funding InformationNational Institutes of Health, National Cancer Institute (CA189477, CA121938, CA155620).
The Cellular Mechanism of Bitter Taste Receptor Mediated Relaxation of Rat Aorta
Authors: Xin Wenkuan , Chen Qian 
Published: November 2023

BackgroundBitter taste receptors, TAS2Rs, are a family of G‐protein‐coupled receptors. TAS2Rs are expressed in mucous epithelial cells of the tongue which mediate the bitter taste and can sense the potential toxins in the food. Recent studies demonstrate that these receptors are expressed in cells outside the gustatory system as well, such as several of smooth muscle tissues. The activation of TAS2Rs can increase the intracellular inositol triphosphate (IP3) thereby the activity of IP3 receptors (IP3R) on the sarcoplasmic reticulum. The Ca2+ that is released through IP3Rs, subsequently activates the large conductance Ca2+‐ and voltage‐activated K+ (BK) channels, and the activation of BK channels leads to the relaxation of smooth muscle.MethodsWe investigated the expression of TAS2Rs in rat aorta using real time qPCR, the functional effect of TAS2Rs activation on rat aortic contractility using isometric tension recordings, and the underlying cellular signaling pathways in smooth muscle cells using confocal microscopy.ResultsqPCR detected the mRNA for TAS2R107 in aorta. The TAS2R agonist, denatonium, relaxed the aorta pre‐contracted with 60 mM K+ in a concentration dependent manner with an EC50 of 215.2 μM (n=6, N=6). The addition of denatonium, as low as 10 μM, caused a transient relaxation of aorta ring pre‐contracted with L‐phynlephrine (10 μM), and the following accumulative addition of denatonium caused a concentration‐dependent further relaxation (n=6, N=6). Denatonium (500 μM) caused a transient increase of electrical field stimulation‐induced (25 Hz‐5 volts) aorta contractions followed by a complete inhibition (n=6, N=6). The confocal microscopy experiments with smooth muscle tissue suggest that the activation of TAS2Rs increased the Ca2+ transient of smooth muscle cells which may underlie the reduction of aorta contractions.ConclusionsWe conclude that the activation of TAS2Rs can reduce the tension of pre‐contracted rat aorta and the TAS2R agonists have the potential implication in the treatment of hypertension.Support or Funding InformationResearch Funding from Southwest University
Functional changes in vascular reactivity to adenosine receptors activation in type I diabetic mice.
Authors: Labazi Hicham , Teng Bunyen , Mustafa S. Jamal 
Published: November 2023

Activation of adenosine receptors (ARs) have been implicated in a number of biological functions including cardiovascular and renal function. Diabetes is known to cause morphological and functional changes in the vasculature, resulting in abnormal responses to various stimuli such as adenosine. The aim for this study was to examine the effect of type I diabetes on the functional changes in conduit (aorta) and resistance arteries (mesenteric arteries, MA) to AR agonist's activation. Diabetes was induced in mice using streptozotocin (STZ, 10mg/Kg/day for 5 days; 12 weeks); concentration response curves (CRC) to acetylcholine, phenylephrine and selective agonists including A1AR (2‐Chloro‐N6‐cyclopentyladenosine, CCPA), A3AR (1‐[2‐chloro‐6‐ [[(3‐iodophenyl) methyl] amino]‐9H‐purin‐9‐yl]‐1‐deoxy‐N‐methyl‐β‐d‐ribofuranuronamide, Cl‐IBMECA), A2AAR (4‐[2‐[[6‐Amino‐9‐(N‐ethyl‐β‐D‐ribofuranuronamidosyl)‐9H‐purin‐2‐yl]amino]ethyl]benzenepropanoic acid hydrochloride, CGS‐21680) and A2BAR (2‐[[6‐Amino‐3,5‐dicyano‐4‐[4‐(cyclopropylmethoxy)phenyl]‐2‐pyridinyl]thio]‐acetamide, BAY 60‐6583) were performed in isolated aortae and MA from STZ and vehicle groups. Diabetes did not affect AR agonists‐mediated relaxation/contraction CRCs in MA. However, aortae from diabetic mice exhibited a significant decrease (p<0.05) in A1R‐mediated vasoconstriction. In addition, the aorta from STZ mice exhibited an increase in phenylephrine‐mediated contraction (EC50 6.88 ± 0.09 in STZ vs. 7.06 ± 0.09 in vehicle; p<0.05), while the relaxation to A2AAR agonist (CGS‐21680) tended to decrease in aortae from the STZ‐treated group, it was not statistically significant. Our data suggest that STZ‐induced diabetes caused functional changes in conduit arteries (decreased A1R‐mediated contraction), and these changes may be a compensatory to counterbalance the increased vascular contractility observed in diabetes.Support or Funding InformationSupported by HL027339