bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of Cytochrome P450 2J2 and Soluble Epoxide Hydrolase in Drug Cardiotoxicity
Authors: Solanki Meetal Satishkumar , Jones Barry , Dawkins‐Hall Laurence , Pointon Amy , Herbert Karl 
Published: June 2021

Cytochrome P450 2J2 (CYP2J2), a multifunctional enzyme that is abundant in cardiac tissue, metabolises endogenous arachidonic acid to epoxyeicosatrienoic acids (EETs) which protect the heart. EETs dilate blood vessels increasing blood flow to heart muscle cells, and reduce inflammation which may protect against development of coronary artery disease. Moreover, it is reported that EETs mitigate the harmful effects of the cancer chemotherapeutic drug, doxorubicin, which causes cardiotoxicity in some patients. In vivo, EETs are rapidly metabolised by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs) which are less biologically active. Several studies have shown that sEH may play an influential role in the development of atherosclerosis and cardiovascular disease, however, its role in cardiotoxicity has not been well reported. The current study aimed to investigate in vitro the role of EETs in the cardiotoxicity of a drug known to interact with CYP2J2, astemizole.Human cardiac myocytes (HCM) and human umbilical vein endothelial cell‐derived EA.hy926 cells, were incubated with astemizole in the absence or presence of inhibitors which were hypothesised to either enhance or diminish EET production. Changes to gene and protein expression were determined and toxicity was assessed.mRNA and protein expression of CYP2J2 and sEH was established in both cell models with higher expression in HCM in line with literature reports. Investigation of sEH expression showed the apparent presence of multiple mRNA isoforms but the predominant isoform was assessed. Treatment of cells with astemizole showed a toxic effect in both cell types accompanied by increased mRNA expression of sEH and CYP2J2 in EA.hy926 cells and HCM. Addition of the sEH inhibitor trans‐4‐[4‐3‐adamantan‐1‐yl‐ureido)‐cyclohexyloxyl]‐benzoic acid, (t‐AUCB), which increases levels of EETs in cells and exogenous 11,12‐EET protected cells against the damaging effects of six hours exposure to astemizole. In response to t‐AUCB in astemizole treated cells sEH expression was increased and CYP2J2 mRNA and protein was decreased; presumably a feedback mechanism exists following sEH inhibition that involves both CYP2J2 and sEH.Taken together, these data show that enhancing EET levels protected against astemizole toxicity in cardiovascular cells in vitro, indicating that the balance between endogenous CYP2J2 and sEH activities may play key role in counteracting damage to the heart by certain drugs. Agents that promote EETs in the heart may be useful adjuncts to counteract the cardiotoxicity due to certain drugs.Support or Funding InformationThis research was supported by Biotechnology and Biosciences Research Council, UK (ref: BB/M503368/1) and AstraZeneca.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A NOVEL INTERACTION BETWEEN AT1 RECEPTOR AND SOLUBLE EPOXIDE HYDROLASE IN DIABETIC RETINOPATHY
Authors: Wang Mong Heng , Al‐Shabrawey Mohamed 
Published: June 2021

There is a critical need to identify new therapeutic targets to treat the microvascular damage associated with diabetic retinopathy (DR) because nearly all patients with type 1 diabetes and 60% of those with type 2 diabetes develop DR. Activation of the renin‐angiotensin system (RAS) contributes to retinal hyperpermeability and neovascularization in DR. Clinical studies have suggested that RAS may be a promising therapeutic target in treating DR. Blockade of RAS has reduced the incidence of DR in clinical studies, yet, RAS blockade did not decrease the incidence of diabetic macular edema or reduce the progression of DR. Hence, the FDA still does not approve the use of RAS blockade to treat DR. In short, lack of understanding of the molecular mechanism of retinal microvascular damage induced by RAS is a critical barrier to the use of RAS blockade to prevent or treat DR. Here we show that 1) Angiotensin II (Ang II) increases retinal soluble epoxide hydrolase (sEH) levels by 2.8 fold (2.84 ± 1.1 vs. 1 ± 0.08, P < 0.05, n = 4), which is blunted by an AT1 blocker; 2) both sEH blockade and knockout (KO), which accumulate epoxyeicosatrienoic acids (EETs), promote Ang II‐induced retinal albumin leakage (an index of blood‐retinal barrier (BRB) injury) by 1.58 (1.58 ± 0.4 vs. 1 ± 0.13, P < 0.05, n = 4) and 2.8 fold (2.8 ± 1.7 vs. 1 ± 0.09, P < 0.05, n = 4), respectively; 3) treatment with synthetic 11,12‐EET potentiates intravitreal Ang II‐induced retinal albumin leakage by 2.2 fold (2.2 ± 0.5 vs. 1 ± 0.18, P < 0.05, n = 4); 4) while sEH KO and blockade reduce Ang II‐induced retinal vascular remodeling, sEH KO causes retinal vascular leakage (fluorescein angiogram analysis) in sEH‐null mice treated with Ang II; and 5) sEH KO potentiates diabetes (STZ)‐induced retinal damage via promoting retinal vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT‐1). Overall, our data demonstrate that in DR, activation of retinal AT1 triggers a compensatory mechanism by increasing retinal sEH, but decreasing EETs. Our studies hold the promise of providing a new strategy, the use of combined EETs blockade with AT1 blocker, to prevent or reduce DR.Support or Funding InformationAHA Grant‐in‐Aid AHASE00144 (M.H.W.) and R01 EY023315 (M.A.S.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increased Podocyte Exosome Release in Glomerular Injury induced by NLRP3 Inflammasome Activation during Hyperhomocysteinemia
Authors: Zhang Qinghua , Li Guangbi , Hong Jinni , Yuan Xinxu , Bhat Owais M. , Lohner Hannah , Li Ningjun , Li Pin‐Lan 
Published: June 2021

The NOD‐like receptor containing pyrin domain 3 (NLRP3) inflammasome has been implicated in podocyte injury and glomerular sclerosis to elevated homocysteine (Hcys) levels in vitro and in vivo. However, it remains unknown how the products of NRLP3 inflammasome activated in cytoplasm are secreted out of podocytes and whether exosome release serves as a critical mechanism to mediate the action of NLRP3 inflammasome activation in Hcys‐induced kidney injury. In the present study, we tried to answer these questions using Smpd1trg/Podocre (a podocye‐specific sphingomyelin phosphodiesterase 1 (Smpd1) transgenic mouse strain) because Smpd1 encodes acid sphingomyelinase that produces ceramide in lysosomes and regulates lysosome function and potentially controls exosome release. It was found that Smpd1trg/Podocre mice had obvious overexpression of Smpd1 gene and remarkable increase in ceramide concentrations in podocytes. These Smpd1trg/Podocre mice were fed a folate‐free (FF) diet to induce hyperhomocysteinemia (hHcys), and ~ 3 folds increase in colocalization of inflammasome proteins, NLRP3 with ASC or with caspase‐1 and ~ 4 folds of IL‐1β production were found in the glomeruli of these FF‐treated Smpd1trg/Podocre mice, suggesting NLRP3 inflammasome formation and activation. Increased IL‐1β production in the glomeruli was accompanied by decreased podocin levels, but enhanced desmin expression in podocytes with glomerular injury as shown by morphological examinations. We also observed significantly increased colocalization of Annexin‐2 and alkaline phosphatase (exosome markers) in glomeruli of FF‐treated Smpd1trg/Podocre mice than their control littermates, indicating the increased exosome release in mice with Smpd1 gene overexpression in podocytes. By Nanoparticle Tracking Analysis, we found that urinary exosomes (60–140 nm in size) were significantly increased especially from 90nm to 140nm in FF‐treated Smpd1trg/Podocre mice, but not in their control littermates. These increased exosomes in urine from FF‐treated Smpd1trg/Podocre mice contained more detectable podocin protein than control littermates, meaning enrichment of podocyte‐derived exosomes in urine from the podocyte‐specific Smpd1 transgenic mice. Taken together, our results suggest that increased ceramide in podocytes due to Smpd1 gene overexpression may increase exosome release and thereby enhance excretion of inflammasome products to induce podocyte dysfunction and glomerular injury.Support or Funding Informationsupported by NIH grants DK54927 to P.L. and, DK107991 to N.L.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thioredoxin Interacting Protein Deficiency Protects Against Obesity‐Induced Podocyte Injury and Glomerular Sclerosis
Authors: Mohammad Razia Sultana , Singh Gurinder Bir , Kshirasagar Naresh , Li Xiang , Hussain Tahir , Li Ningjun , Li Pin‐Lan , Koka Saisudha , Boini Krishna M. 
Published: June 2021

Recently we have shown that activation and formation of NLRP3 inflammasomes in podocytes is implicated in the development of obesity‐induced glomerular injury. Moreover, we demonstrated that NADPH oxidase dependent O2•− production (ROS) mediates the obesity‐induced NLRP3 inflammasomes activation and thereby results in podocyte injury and glomerular damage. However, it remains unknown how NADPH oxidase‐derived ROS is sensed by NLRP3 inflammasomes in podocytes upon visfatin stimulation. The current study tested whether thioredoxin‐interacting protein (TXNIP) mediates obesity‐induced NLRP3 inflammasome activation and consequent glomerular injury. In cultured podocytes, confocal microscopic analysis showed that visfatin treatment significantly increased the colocalization of NLRP3 with Asc or caspase‐1 in podocytes compared to control cells. Pretreatment with TXNIP siRNA abolished the visfatin‐induced inflammasome formation. Correspondingly, visfatin treatment significantly increased the caspase‐1 activity and IL‐1β production compared to control cells. The TXNIP siRNA transfection significantly attenuated the visfatin‐induced caspase‐1 activity and IL‐1β production. Further immunofluorescence analysis showed that visfatin treatment significantly decreased the podocin expression (podocyte damage) compared to control cells. However, pretreatment with TXNIP siRNA attenuated the visfatin‐induced podocin reduction. In in vivo studies, adult C57BL/6J male mice were fed a high fat diet for 14 weeks to induce obesity, and TXNIP was inhibited by verapamil (1 mg/ml in drinking water). High fat diet significantly increased the plasma visfatin levels compared to control mice. In addition, RT‐PCR analysis showed that high fat diet significantly increased the Nlrp3 expression in glomeruli of wild type mice compared to control mice but not in verapamil treated mice. Furthermore, TXNIP inhibition significantly reduced IL‐1β production in glomeruli of mice with obesity. Correspondingly, TXNIP inhibitor, verapamil attenuated obesity‐induced proteinuria, glomerular damage and podocyte injury. In conclusion, our results demonstrate that TXNIP binding to NLRP3 is a key signaling mechanism necessary for obesity‐induced NLRP3 inflammasome formation and activation and subsequent glomerular injurySupport or Funding InformationNIH grant, DK104031This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐liver fibrotic effect of triggering receptor expressed on myeloid cells 2 (TREM2) via macrophage phenotype switching
Authors: Kang Byeongseok , Kim Nayoun , Yang Jin Won , Kang Keon Wook 
Published: June 2021

Macrophage phenotype switching is critical for the progress of inflammatory diseases. The aim of this study is to investigate the potential role of triggering receptor expressed on myeloid cells‐2 (TREM2) in macrophage phenotype switching and liver fibrosis. TREM2 protein expression was increased in fibrotic livers from human and mouse. Transforming growth factor‐β1 (TGFβ1), a representative stimulus for the differentiation of M2c macrophage, specifically increased TREM2 expression in Raw264.7 macrophage cells. Co‐culture of hepatic stellate cells (HSCs) with TREM2‐overexpressing Raw264.7 cells showed the decreased expression of IL‐6 compared to co‐culture of HSCs and mock‐Raw264.7 cells. TREM2‐transgenic mice showed the decreased liver fibrosis in CCl4‐induced liver fibrosis model, whereas TREM2‐null mice showed the aggravated liver fibrosis compared to wild‐type mice. The basal mRNA level of interleukin‐10 was enhanced and the basal level of monocyte chemoattractant protein‐1 was decreased in liver samples from TREM2‐transgenic mice. Therefore, the overexpressed TREM2 in macrophages from fibrotic liver suppresses the progress of hepatic fibrogenesis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Mechanistic Effects of JNK and p53 Knockdown on Hepatocyte Viability Following Dipyridyl Herbicide Exposure
Authors: Nguyen Ngoc U. , Stamper Brendan 
Published: June 2021

ObjectiveThe objective of this study is to characterize key pathways that contribute to the hepatotoxic effects of two dipyridyl herbicides, paraquat and diquat.MethodsDose‐dependent toxicity assays were performed in an in vitro liver model (TGF‐alpha transgenic mouse hepatocytes; TAMH) to determine LC50 values for paraquat and diquat. Transient knockdown of JNK and p53 was performed using siRNA. Target knockdowns were confirmed using protein immunoblotting. Cell viability assays were then conducted to determine how each transfection affected cell survival following exposure to either paraquat or diquat.ResultsLC50 values were determined to be 57μM and 18μM for paraquat and diquat, respectively. siRNA‐based knockdown decreased JNK protein expression by 31%. This correlated with significant increases in cell viability for transfected cells exposed to either paraquat (21%) or diquat (15%). siRNA‐based knockdown decreased p53 protein expression by 66%. This correlated with a significant but small decrease in the viability of transfected cells exposed to paraquat only (6%).ConclusionExperimental LC50 values revealed diquat to be more hepatotoxic than paraquat in the TAMH line. The siRNA‐based knockdown of JNK provides evidence that JNK plays a crucial role in propagating toxicity caused by these herbicides. In contrast, effects from the p53 knockdown experiments had either no effect (diquat exposure) or propagated toxicity to a small but significant extent (paraquat). These observations suggest affecting targets that precede p53 during apoptotic signaling are crucial to rescuing hepatocytes from the toxic effects of dipyridyl herbicides.Support or Funding InformationThis work was supported by the Pacific University Research Incentive Grant Program and an ASPET Summer Undergraduate Research Fellow (SURF) Individual Award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altered Steroid and Drug Metabolism by a Cytochrome P450 Oxidoreductase Variant Found in Apparently Normal Population
Authors: Pandey Amit V. , Parween Shaheena , Roucher‐Boulez Florence , Morel Yves 
Published: June 2021

Background and AimsA broad spectrum of human diseases, including abnormalities in steroidogenesis, are caused by mutations in the cytochrome P450 oxidoreductase (POR) (1–3). POR transfers electrons from NADPH to small molecules, non‐P450 redox partners and microsomal cytochrome P450 proteins. Our aim was to check if POR variations from non‐clinical samples (1000 genomes database), can be disruptive. The Y607C variant of POR (rs72557954, NM_000941.2:c. 1820A>G) has been reported in population studies and is prevalent in South Asians (4), but was predicted to be likely pathogenic. We performed detailed enzymatic and biochemical characterizations of Y607C variant of POR to study its effect on different substrate and redox partners.MethodsWe analysed the ability of POR wild type (WT) and Y607C variant to reduce ferricyanide, MTT, cytochrome c and drug, and steroid metabolizing cytochrome 450 enzymes. The POR WT and Y607C were expressed and produced as recombinant proteins while CYP19A1 and CYP3A4 were produced as His‐tagged recombinant proteins and purified by affinity chromatography. The effect of mutation on cofactor (FAD/FMN) binding and activity under varying substrate and cofactor conditions was performed.ResultsWe found varied effects of Y607C mutation on activities with different partners. As compared to WT, Y607C variant showed 66% cytochrome c and 91 % ferricyanide reduction activity but had only 13 % MTT reduction activity. The POR Y607C did not affect flavin content but NADPH binding was severely affected. With varying NADPH concentration, Y607C showed ~95% decrease in supporting CYP19A1 and CYP3A4 activity. This mutation was later identified in patients with POR deficiency.ConclusionsIdentification of severe effects of this mutation on both drug and steroid metabolizing cytochrome P450s indicates that likely pathogenic mutations may be found in apparently normal (non‐clinical) population. Their combination as compound heterozygotes or homozygous may lead to severe impact on both steroid and drug metabolism by modification of POR redox partner activities. Variations in POR need to be evaluated individually. Most importantly, advanced identification of disease causing variants in POR will help in understanding the POR deficiency in patients if the same mutations are later identified.Support or Funding InformationThis research was funded by grants to Amit V Pandey from the Swiss National Science Foundation (31003A_134926).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
UGT1A10 drives tissue‐specific differences in the clearance of the chiral tobacco carcinogen, NNAL
Authors: Kozlovich Shannon , Chen Gang , Lazarus Philip 
Published: June 2021

Tobacco‐specific nitrosamines (TSNAs) are among the most potent carcinogens found in tobacco, with 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) the most potent and one of the most abundant. NNK is extensively metabolized to the equally carcinogenic chiral 4‐ (methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL). (S)‐NNAL exhibits higher stereoselective tissue retention in mice and humans and has been shown to be more carcinogenic in mice than its enantiomeric counterpart, (R)‐NNAL. This differential carcinogenic potential makes it increasingly important to know which UGT enzymes target specific NNAL enantiomers for glucuronidation. NNAL is detoxified primarily by four hepatic UDP‐glucuronyltransferase (UGT) enzymes: UGTs 1A9, 2B7, 2B10 and 2B17; each of these UGTs have been shown to exhibit different stereo‐specificity for the formation of glucuronides of the different NNAL enantiomers. The goal of the present study was to identify the UGTs involved in extra‐hepatic NNAL metabolism and examine differences in the stereospecific glucuronidation of NNAL between tissue types with different UGT expression. Large differences in stereo‐specificity for (S)‐ versus (R)‐NNAL were identified upon comparison of NNAL‐glucuronides (Gluc) formed in reactions of extra‐hepatic UGT1A10‐over‐expressing cell microsomes upon incubation with racemic NNAL, with an enantiomeric preference for (R)‐NNAL as a substrate when compared to (S)‐NNAL. When UGT1A10‐over‐expressing cell microsomes were incubated with pure NNAL enantiomers, large difference in kinetics were observed, with a Km=1.5 mM and Vmax=4.6 pmol/min/mg for (R)‐NNAL, vs a Km>16 mM and Vmax=1.3 pmol/min/mg for (S)‐NNAL. Human liver microsomes preferentially formed (S)‐NNAL‐Gluc while microsomes from human intestine, larynx, esophagus, tonsil, floor of mouth, and tongue all preferentially formed (R)‐NNAL‐Gluc upon incubation with racemic NNAL. Liver and intestine were shown to express UGTs 1A9, 2B7, and 2B17 while intestine, unlike liver, has also been shown to express UGT1A10. All other tissue types analyzed were shown to express only UGTs 1A10 and 2B17, with the exception of esophagus that also expressed UGT2B7. These data indicate that UGT1A10 may drive the difference in differential tissue‐specific clearance of the specific NNAL enantiomers known to induce tobacco‐related cancers.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bioengineering of single ncRNA molecule for multi‐targeting against NSCLC
Authors: Petrek Hannah Elizabeth , Ho Pui Yan , Zhang Qianyu , Batra Neelu , Jilek Joseph , Yu Ai‐Ming 
Published: June 2021

Lung cancer is the second most common cancer among both men and women in the United States. The majority of lung cancer cases are classified as non‐small cell lung cancer (NSCLC). Current NSCLC treatments include resection surgery, radiation, and pharmacotherapy which all provide some benefits and have limitations. As a result, lung cancer caused deaths account for about 27% of all cancer deaths in the US. Therefore, new therapeutic agents are highly demanded for NSCLC. MicroRNAs (miRNA or miR) are a class of genome‐derived, small noncoding RNAs (ncRNA) in cells that govern target gene expression through mRNA degradation or translational inhibition. Some miRNAs such as miR‐34a, miR‐124 and let7c are commonly downregulated in NSCLC tissues/cells and target various oncogenes such as CDK6, STAT3, and Ras. Restoring the expression or function of such dysregulated, tumor suppressive miRNAs represents a new strategy for cancer therapy. Aiming at simultaneously introducing multiple miRNAs in NSCLC cells for multi‐targeting, we employed our recently developed ncRNA bioengineering technology to produce a single ncRNA molecule bearing multiple miRNAs. Our data showed that multi‐targeting ncRNA agents were successfully expressed in E. coli. We were able to purify recombinant ncRNAs to a high degree of homogeneity using anion exchange FPLC methods. We further demonstrated that treatment of human NSCLC cells with bioengineered ncRNAs elevated cellular miR‐34a, miR‐124 and let‐7c levels, reduced protein levels of target genes including CDK6, STAT3 and Ras, and consequently inhibited NSCLC cell proliferation. Therefore, using single ncRNA agent for multi‐targeting may be more effective for the treatment of NSCLC.Support or Funding InformationThis study was supported by grants R01GM133888 and U01CA175315 from NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.