该【A Quantitative HILIC–MS MS Assay of the Metabolic Response of Huh-7 Cells Exposed to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Qing Liu 】是由【探春文档】上传分享，文档一共【14】页，该文档可以免费在线阅读，需要了解更多关于【A Quantitative HILIC–MS MS Assay of the Metabolic Response of Huh-7 Cells Exposed to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Qing Liu 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ectsofTCDDincludeadaptiveresponses,wastingsyndrome,tumorpromotion,immunotoxicity,developmentaltoxicity,oxicity[10–14].Inmostcases,bio?uidsortissuesfrominvivoexperimentshavebeenanalyzedviametabolomics[15,16],butthedevelopmentofrobustandhigh-throughputpro?lingmethodsofintracellularmetabolitesusingcellculturesystems[17,18]?cationremainhighlychallengingtasksduetothedi erentphysicochemicalpropertiesofmetabolites,theirbroadrangeofconcentration(fMtomM),andtheparedi ,wereportahydrophilicinteractionliquidchromatography(HILIC)–ultrahigh-pressureliquidchromatography(UHPLC)–MS/MSapproachpermittingthefast,selective,andquantitativemeasurementofpolarmetabolitesinvolvedincriticalmetabolicpathwaysin?,uratelyquanti?edincludingaminoacids(27),organicacids(14),sugars(2),phosphorylatedsugars(7),nucleobases(12),nucleotides(19),phosphorylatedmetabolites(11),hydrophilicvitamins(8),andcoenzymes(7).TheHILICmethodwasoptimizedbasedontheLCgradient,columntemperature,ionconcentration,(CYP1A1)inHuh-7cells[19].TocharacterizethetoxicityresponsetoTCDD,theHILIC–UHPLC–MS/MSmethodwasusedtoexamineextractsfromHuh-(PLS-DA)identi?eddi ,1HNMR-basedplementarymetabolicperspectiveinordertovalidatetheUHPLC–HILIC–MS/?,ponentsofcentralcarbon,aminoacid,andnucleotidemetabolismwerequantitatedbythedevelopedHILIC–MS/,theHILIC–MS/MSmethodwasvalidatedwithHuh-7cellsfortheglycolyticpathwayandthenappliedonTCDDtoxicologywithHuh--UHPLC-MS/MSMethodMRMtransitions(MT),retentiontime(RT),linearrange(LR),limitofdetection(LOD),andlimitofquantitation(LOQ)poundsfromdi %M,respectively,ontheWatersXevoTQDmassspectrometer(Milford,MA,USA).PhosphorylatedcompoundssuchasE4P(LOD=M,LOQ=M),F1,6BP(LOD=M,LOQ=M),R5P(LOD=M,LOQ=M),Ru5P(LOD=M,LOQ=M),anicacidorcitricacid/isocitricacid(LOD=M,LOQ=M)andshikimicacid(LOD=M,LOQ=M)showedrelativelyhigherLOD/LOQvaluesowingtotheirlowionizatione(N=5)ofthespike-%%,with84%ofRSDbelow10%,andtheinterdayprecision(N=5)%to:..Metabolites2019,9,%,with83%ofRSDbelow15%.-quarter(23%)ofpounds,andfortheremainder(77%)–UHPLC–MS/MSmethodisrelativelyeasytotransfertootherplatforms,includingtheWatersXevoTQS,,glutamine/lysine,F6P/G6P,andthesemetabolitescannotbedi erentiatedbyMS/,uratequanti?cation[20].Moreover,–phasetransferofanalytesbetweenanaqueousstationarysolventandrelativelynon-anicmobilephase[21–23].HILICalsoinvolvespolarinteractions(hydrogenbonding),LCconditionswereoptimizedbasedonMaliketal.[24](TableS3).Mobilephaseionicstrength,temperature,andstationaryphaseessfulHILIC[25].Here,thein?uenceofmobilephasegradient,ionicstrength,andcolumntemperaturewereevaluated(FigureS1).Twelvemetabolitesfromdi erentclasseswereselected,includingniacinamide,hippuricacid,creatinine,valine,proline,VitB12,FAD,malicacid,acetylCoA,AMP,R5P,-mode,andtherestwereacquiredinESI+?vepairsofisomersisshowninFigureS2;the?vepairsofisomersachievebasicseparation(R1)inourstudy,-lycolysisPathwayAfterestablishingandoptimizingtheHILICmethod,weaimedtoquantifypolarmetabolitesinHuh-7cellstreatedwithbromopyruvate,-BrPy[26–28],ahalogenatedformofpyruvate,hashighanti-tumoractivityasaglycolyticinhibitor,bothinvitroandinvivo[29].Theprimarytargetof3-BrPyinglycolysisappearstobeglyceraldehyde-3-phosphatedehydrogenase(GAPDH).Normally,hexokinaseisoformII(HKII)[30]isnotexpressedinmosttissuesbutishighlyexpressedinmanytumorcellsincludinghepatocellularcarcinoma(suchasHuh-7cells,HepG2cells).Unlikeotherchemotherapeuticagents,3-BrPyislesstoxictocells[26].Ganapathy-Kanniappanetal.[31]foundthat3-BrPysuppressedtheproliferationofHep3Bcellsat150.[32]showedthattheshort-term(24–48h)lutamineresultedingrowtharrest,,Huh-7cellsweretreatedwith3-BrPyatconcentrationsof5M,15M,50M,and100M,andmediawithlowglucose(2mM)wasusedasthepositivecontrol(allthemediawassupplementedwith1%sodiumpyruvate).PharmacologictargetingofaerobicglycolysisinHuh-(FigureS4A),theexposureofHuh-7cellsfor24hto5–100Mof3-BrPydidnotcauseobviouscelldeath,whichissimilartothatreportedfromprimaryratastrocytes[33].Eightymetabolitesweredetectedandquanti?edusingourmethod(TableS5).ponentsanalysis(PCA)ofpolarmetabolitesofHuh-7cellstreatedwithdi erentconcentrationsof3--?cally,thequantitationofimportantmetabolitesinvolvedinglycolysiswasalsoanalyzedamonggroupsusingone-wayANOVA(FigureS5).Asexpected,intracellularATPandNADHsigni?cantlydecreasedinadose-dependentmannerafteradding3-,F1,6-BP,and3-phosphoglycericacidintheglycolysispathwaydecreasedaftertreatmentwith50Mand100Mof3-BrPy,phosphoenolpyruvatedecreasedaftertreatmentwith100Mof3--7cellswasinhibitedby3-:..Metabolites2019,9,1184of14PKM2(pyruvatekinase)umulatephosphoenolpyruvate,whichprovidesprecursorsforthesynthesisofaminoacids,nucleicacids,andlipids,thusprovidingcancercellswithagrowthadvantage[34].TheintermediateproductsintheTCAcycleshowednosigni?cantchangein3-BrPytreatment,indicatinglittlein?uenceonTCAby3-BrPywithaconcentrationbelow100;thedecreaseofglutamineindicatedtheweakenedactivityinuptakeduetoglycolysisblockage[35].Glucogenicaminoacidsynthesisincludingalaninedecreased,whileketogenicaminoacidleucineincreasedinthisMetabolites2019,9,(GSH)andglutathioneoxidized(GSSG)hadnosigni?cantchangesinourresearch,suggestingthatunlikeastrocytes[influenceonTCAby3-],3-BrPymaynotdepletetheGSHofHuh-,glycolysisinHuh-7cellswasinhibitedby3-BrPy,indicatingthatthetwoimportantnutrientsforproliferatingcells;thedecreaseofglutamineindicatedtheweakenedactivityinuptakeduetoglycolysisblockage[35].GlucogenicaminoacidsynthesisincludingalanineUHPLC–HILIC–MSdecreased,,thismethodwasappliedtounderstandlow-doseTCDDtoxicity.(GSH)andglutathioneoxidized(GSSG)hadnosignificantchangesinourresearch,suggestingthatunlikeastrocytes[33],3-BrPymaynotdepletetheGSHofHuh-,-7CellsglycolysisinHuh-7cellswasinhibitedby3-BrPy,indicatingthattheUHPLC–HILIC–MS/,thismethodwasappliedtounderstandlow-,particularlyintermsofitshepatotoxic,carcinogenic,-7Cells ects[36,37],[8,19](usuallyTCDDhasbeenwidelystudiedinvivoandinvitro,particularlyintermsofitshepatotoxic,approximately10nMto10,000nM),butrelativelyfewstudieshaveusedmetabolomics,particularlyatcarcinogenic,andphysiologicaleffects[36,37],
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