ReportforASEA:Datafromaninvitrostudyrelatingtoproductsafety

StudyPerformedbyPacificNorthwestNationalLaboratory(PNNL)*
PurposeandDisclaimer:
ThisdataistakenfromaninvitroscientificstudydonetodeterminethebioactivityofASEAon eukaryoticcellsinacontrolledenvironment.Thisreportoffersasummaryoftheresultsrelatedto safetydataforASEAonacellularlevel.Thisdataanswersthequestion:Isthereanyevidenceofatoxic responseifASEAcomesintocontactwitheukaryoticcells?

Explanationoftranscriptionfactoractivationscience:
Whenacellisstressedbyatoxin,thecellrespondsbysendingacertainsetoftranscriptionfactors intothenucleus.Onceinsidethenucleus,thesetranscriptionfactorsactivatethegenesresponsiblefor cellulardefenseandprotectionagainsttoxins(suchastheinflammatoryresponse).Thetranslocationof certaintranscriptionfactorsintothenucleuscanbeseenunderafluorescentmicroscopewhenthecells stainedbyspecificindicatordyes. Ifthecellundergoesatoxicresponse,thefluorescentdyeispulledintothenucleusalongwiththe transcriptionfactor.Inthisexperimenttwotranscriptionfactors,thep65subunitofNFkappaBandP Jun,weremonitored.Thesetwotranscriptionfactorsareknowntobeactivatedinalltoxicresponses. Inthephotographsfromthefluorescentmicroscopicimagesofthecells,atoxicresponseisregisteredif thegreendyeisseentomoveintothenucleus.

Procedure:
Thetargeteukaryoticcellswereculturedindishesandexposedrespectivelyto(1)Phosphate BufferedSaline(PBS)thenegativecontrol(notoxicresponseexpected),(2)5%ASEAEquivalentto replacing5%ofthenutrientsolution(bloodplasma)withASEA,(3)20%ASEAEquivalenttoreplacing 20%ofthenutrientsolutionwithASEA,and(4)Aknowntoxinthepositivecontrol(toxicresponse expected). Theresponseofthetranscriptionfactors,thep65subunitofNFkappaBandPJun,were photographedunderamicroscopeafterexposuretothefoursolutionslistedabove.ADAPIstainwas alsoappliedtothenucleiinordertohelpcomputersoftwaretofindthenucleusinthepictures.The softwareautomaticallytalliedtheamountofdyeinthenuclei.InthecaseofPJun,measurementsof overonehundredscellsweremadeinordertocompilethesummarychart.

*PNNLdoesnotinanywayendorseASEA,itmerelyactedasanindependentlaboratoryinperforming andproducingthesetestresults.

 Results: P65/NFkappaB
Theimagescellswithstainedp65subunitofNFkappaBareshownbelow.Itisvisuallyevidentthat notoxicresponseisregisteredforexposureofthecellstoASEAcomparedtotheclearpositiveresponse isseenintherightmostpositivecontrol.Thep65subunitremainsontheoutsideofthenucleiinthe leftmost3pictures,indicatingthatnotoxicresponseisregisteredbythecells.

PBS5%ASEA20%ASEAKnownToxin

*PNNLdoesnotinanywayendorseASEA,itmerelyactedasanindependentlaboratoryinperforming andproducingthesetestresults.

 PJun
AsreinforcementoftheNFkappaBresults,thePJundataalsoshowsvisualevidencethatnomajor toxicresponseregisters.ItwasnecessaryforthePJundatatobeaveragedovermorethan100cellsin ordertogetstatisticallysignificantnumericalresults.Theresultingchartclearlyshowsthatnotoxicity existsforthe5%ASEA.Bloodconcentrationfororaldoses,however,willnevergetanywhereneareven 1%.

PBS5%ASEA20%ASEAKnownToxin

60

Nuclear P-Jun Levle (RFU/nucleus)

None
40

PBS 5%ASEA

20

20%ASEA KnownToxin

Conclusions
DirectexposureofcellstorelativelyhighconcentrationsofASEAdoesnotregisterasignificanttoxic responseasmeasuredbyNFkappaBandPJunnucleartranslocation.Basedontheseresults,ASEA, orallyadministered,shouldnotmanifestatoxicresponseorinflammationtoanyoftheexposedtissues.

*PNNLdoesnotinanywayendorseASEA,itmerelyactedasanindependentlaboratoryinperforming andproducingthesetestresults.