cellular and molecular response to nanoparticle exposure by: jewels morgan
TRANSCRIPT
Cellular and Molecular Cellular and Molecular response to response to Nanoparticle ExposureNanoparticle Exposure
By: Jewels MorganBy: Jewels Morgan
What is a What is a nanoparticle?nanoparticle? Small particles with a dimension Small particles with a dimension
of less than 100nmof less than 100nm
Nanoparticles UsesNanoparticles Uses
Used in many different Used in many different applications applications – BiomedicalBiomedical– OpticalOptical– ElectronicsElectronics– Hygiene ProductsHygiene Products– Safety ProductsSafety Products– Food IndustryFood Industry
Cross biological membraneCross biological membrane Small surface area/volume ratioSmall surface area/volume ratio Toxicity to human tissues and cell Toxicity to human tissues and cell
culturescultures Possible DNA interactions, damage, Possible DNA interactions, damage,
mutationsmutations Uptake by mitochondria and possible Uptake by mitochondria and possible
damagedamage Cell deathCell death
What are possible What are possible dangers of Nanoparticle dangers of Nanoparticle Exposure?Exposure?
Previous ResearchPrevious Research
Cultured two different cell linesCultured two different cell lines– CHO (Chinese Hamster Ovary)CHO (Chinese Hamster Ovary)– COS-7 (African Green Monkey COS-7 (African Green Monkey
Kidney Cells)Kidney Cells) Morphology of both cell linesMorphology of both cell lines Cytotoxicity of both cell lines Cytotoxicity of both cell lines Bead location Bead location Preliminary ApoptosisPreliminary Apoptosis
Particles UsedParticles Used
Latex Beads, Blue dyedLatex Beads, Blue dyed Ultra fine nanoparticles (55nm)Ultra fine nanoparticles (55nm) Fine particles (800nm)Fine particles (800nm)
Fall 2007 Research Fall 2007 Research ResultsResults Cytotoxicity ResultsCytotoxicity Results
– CHO 24 hrsCHO 24 hrs– COS-7 48 hrsCOS-7 48 hrs
Both bead sizes showed cytotoxic affects to cellsBoth bead sizes showed cytotoxic affects to cells MorphologyMorphology
– Cultured cells morphology matched ATCC Cultured cells morphology matched ATCC databasedatabase
Nanoparticle LocationNanoparticle Location– Determined localization of the Determined localization of the
nanoparticles nanoparticles to be in the cytoplasmic regionto be in the cytoplasmic region
Pictures of Research Pictures of Research ResultsResults
CHO 24 hr exposure
Bead Location
Morphology
Current Research Spring Current Research Spring 20082008
ApoptosisApoptosis– CHO cellsCHO cells– CASP1 (Caspase)CASP1 (Caspase)
Chromosomal Spread-AberrationsChromosomal Spread-Aberrations– Halted in metaphase using colchicineHalted in metaphase using colchicine
Gene Expression of Pro-Inflammatory Gene Expression of Pro-Inflammatory Cytokines-PCR RNACytokines-PCR RNA– IL-6IL-6– IL-18IL-18
Protein ConcentrationProtein Concentration
ApoptosisApoptosis
Programmed Cell deathProgrammed Cell death CHO Cells CHO Cells
– Treated for 24 hrs with 1Treated for 24 hrs with 1μμL of beadsL of beads– Isolated DNAIsolated DNA– Verified purity and concentrationVerified purity and concentration
SpectrophotometerSpectrophotometer λλ260nm and 260nm and λλ280nm 280nm
TreatmentTreatment DNA DNA ConcentrationConcentration
DNA DNA λλ260nm/280nm 260nm/280nm
RatioRatio
Control 1Control 1 .635.635μμg/g/μμLL 1.891.89
Control 2Control 2 .945 .945 μμg/g/μμLL 2.122.12
55nm beads 155nm beads 1 .650 .650 μμg/g/μμLL 1.881.88
55nm beads 255nm beads 2 .280 .280 μμg/g/μμLL 1.861.86
800nm beads 1800nm beads 1 .635 .635 μμg/g/μμLL 1.441.44
800nm beads 1800nm beads 1 .880 .880 μμg/g/μμLL 1.611.61
DNA PurityDNA Purity
All values determined using a 1:100 dilutionRatio value 1.60 to 1.80=pure DNA
DNA Analysis following DNA Analysis following nanoparticle treatmentnanoparticle treatment
Showed no Showed no fragmentation of fragmentation of DNADNA
No significant No significant evidence of evidence of Apoptosis Apoptosis occurringoccurring
Ladder 55nm
CHO DNA
Pro-Inflammatory Pro-Inflammatory CytokinesCytokines Isolated RNAIsolated RNA
– TRI ReagentTRI Reagent Lyse cells to release RNALyse cells to release RNA
– RNA Isolated from Controls, 55nm, & RNA Isolated from Controls, 55nm, & 800nm exposure cells800nm exposure cells
– PCR samples with designed primers for:PCR samples with designed primers for: IL-6IL-6 IL-18IL-18 CaspaseCaspase Beta Actin Beta Actin
IL-6IL-6– Released following oxidative stress Released following oxidative stress
to cell to cell IL-18IL-18
– Released following oxidative stress Released following oxidative stress to cellto cell
CASPASECASPASE– Elevated during Apoptosis Elevated during Apoptosis
Pro-Inflammatory Pro-Inflammatory Cytokines and Cytokines and Apoptotic geneApoptotic gene
Primer DesignPrimer Design
NCBI Database NCBI Database – Chinese Hamster Chinese Hamster (Cricetulus griseus) (Cricetulus griseus)
IL-6IL-6– Mouse (Mouse (Mus musculus) Mus musculus) Caspase -1Caspase -1
Unknown Sequence in CHOUnknown Sequence in CHO
– Mouse (Mouse (Mus musculus) Mus musculus) IL-18IL-18 Unknown Sequence in CHOUnknown Sequence in CHO
– Chinese Hamster (Chinese Hamster (Cricetinae gen. sp) Cricetinae gen. sp) Beta ActinBeta Actin Endogenous gene expressionEndogenous gene expression
Primer Design cont.Primer Design cont.
Primers need to have:Primers need to have:– 50% or greater gc content50% or greater gc content– Annealing temperature ~60.0Annealing temperature ~60.0ºCºC– Product size ~350-450bpProduct size ~350-450bp
Primers designed using Primer 3 Primers designed using Primer 3 software after importing software after importing sequence from NCBI databasesequence from NCBI database
Primer size 20bpPrimer size 20bp
RNA Purity and RNA Purity and Concentration ResultsConcentration Results
TreatmentTreatment RNA RNA ConcentrationConcentration
RNA RNA λλ260nm/280nm 260nm/280nm
RatioRatio
Control 1Control 1 .572.572μμg/g/μμLL 2.072.07
Control 2Control 2 .136 .136 μμg/g/μμLL 1.481.48
55nm beads 155nm beads 1 .656 .656 μμg/g/μμLL 2.822.82
55nm beads 255nm beads 2 .144 .144 μμg/g/μμLL 2.002.00
800nm beads 1800nm beads 1 .202 .202 μμg/g/μμLL 1.801.80
800nm beads 1800nm beads 1 .164 .164 μμg/g/μμLL 1.781.78
All values determined using a 1:100 dilutionRation value of 1.8-2.0=Pure RNA
Gene ExpressionGene Expression
CHO IL-6 gene
Ladder
Ladder
CHO CAS gene
Ladder
1000bp
500bp
Con 55nm 800nm
1000bp
500bp
Con 55nm 800nm
Pro-Inflammatory Pro-Inflammatory Cytokine Response Cytokine Response cont.cont.
Ladder
Control 55nm 800nm
IL-18 Beta Actin
Ladder Control 55nm 800nm
Chromosomal SpreadsChromosomal Spreads
Chromosomal Spread-Chromosomal Spread-AberrationsAberrations
Control CHO Chromosomes 55nm Chromosome Spread 800nm Chromosome Spread
* Significantly different from control student’s t-test
Chromosome Spread Analysis
0
2
4
6
8
10
12
14
16
Control 55 nm 800 nm
Treatments
Nu
mb
er
of
ch
rom
oso
mes *
Protein QuantificationProtein Quantification
TreatmentTreatment Protein ConcentrationProtein Concentration
Control 1Control 1 .750mg/mL.750mg/mL
Control 2Control 2 .765mg/mL.765mg/mL
55nm beads 155nm beads 1 .840mg/mL.840mg/mL
55nm beads 255nm beads 2 .910mg/mL.910mg/mL
800nm beads 1800nm beads 1 .905mg/mL.905mg/mL
800nm beads 2800nm beads 2 .909mg/mL.909mg/mL
SummarySummary
DNA ApoptosisDNA Apoptosis– No detection of Apoptotic bandsNo detection of Apoptotic bands– Increase in Caspase expressionIncrease in Caspase expression
Cytokine ExpressionCytokine Expression– IL-6 increased following treatment with 55nm IL-6 increased following treatment with 55nm
beads compared to controlbeads compared to control– IL-18-No detection of gene expressionIL-18-No detection of gene expression
Slight protein concentration in treated Slight protein concentration in treated samplessamples
Chromosome number and appearance Chromosome number and appearance altered following treatmentaltered following treatment
Special Thanks to….Special Thanks to…. Dr. Jacqueline Jordan-Research MentorDr. Jacqueline Jordan-Research Mentor Truyen Derek Pham-Supplies DonationTruyen Derek Pham-Supplies Donation
– Emory UniversityEmory University Sheryl Sands-Lab TechnicianSheryl Sands-Lab Technician Dr. Michelle FurlongDr. Michelle Furlong CSU Natural Sciences Faculty & StaffCSU Natural Sciences Faculty & Staff