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
None
Nuclear P-Jun Levle
(RFU/nucleus)

40 PBS

5%ASEA

20 20%ASEA

KnownToxin

Conclusions

DirectexposureofcellstorelativelyhighconcentrationsofASEAdoesnotregisterasignificanttoxic
responseasmeasuredbyNFkappaBandPJunnucleartranslocation.Basedontheseresults,ASEA,
orallyadministered,shouldnotmanifestatoxicresponseorinflammationtoanyoftheexposedtissues.

*PNNLdoesnotinanywayendorseASEA,itmerelyactedasanindependentlaboratoryinperforming
andproducingthesetestresults.