Asea Safety Red Ox Signaling Molecules

8/6/2019 Asea Safety Red Ox Signaling Molecules

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*PNNLdoesnotinanywayendorseASEA,itmerelyactedasanindependentlaboratoryinperforming

andproducingthesetestresults.

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.
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2/3



Results:

P65/NFkappaB

Theimagescellswithstainedp65subunitofNFkappaBareshownbelow. Itisvisuallyevidentthat

notoxicresponseisregisteredforexposureofthecellstoASEAcomparedtotheclearpositiveresponse

isseenintherightmostpositivecontrol. Thep65subunitremainsontheoutsideofthenucleiinthe

leftmost3pictures,indicatingthatnotoxicresponseisregisteredbythecells.

PBS 5% ASEA 20% ASEA Known Toxin
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3/3



PJun

AsreinforcementoftheNFkappaBresults,thePJundataalsoshowsvisualevidencethatnomajor

toxicresponseregisters. ItwasnecessaryforthePJundatatobeaveragedovermorethan100cellsin

ordertogetstatisticallysignificantnumericalresults. Theresultingchartclearlyshowsthatnotoxicity

existsforthe5%ASEA. Bloodconcentrationfororaldoses,however,willnevergetanywhereneareven

1%.

PBS 5% ASEA 20% ASEA Known Toxin

Conclusions

DirectexposureofcellstorelativelyhighconcentrationsofASEAdoesnotregisterasignificanttoxic

responseasmeasuredbyNFkappaBandPJunnucleartranslocation. Basedontheseresults,ASEA,

orallyadministered,shouldnotmanifestatoxicresponseorinflammationtoanyoftheexposedtissues.

0

20

40

60

NuclearP-Ju

nLevle

(RFU/nucleus)

None

PBS

5%ASEA

20%ASEA

KnownToxin
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Documents

Asea Safety Red Ox Signaling Molecules