In Vitro Assessment of Nano Material Toxicity
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Transcript of In Vitro Assessment of Nano Material Toxicity
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I n vitro assessment of
Nano Mater ial
Toxicity
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Introduction
Nanomaterialsare generally in the 1-100 nm rangeand can be composed of many different base
materials (carbon, silicon, and metals such as gold,cadmium, and selenium).
Nanomaterials also have different shapes: referred toby terms such as nanotubes, nanowires, crystallinestructures such as quantum dots, and fullerenes.
Nanomaterials often exhibit very different propertiesfrom their respective bulk materials: greater strength,conductivity and fluorescence.
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Major Advantages ofIn Vitro ASSESSMENT
Reduction in animal use
Greater experimental control over chemical
concentrations, environmental composition Uniform biological systems (cell lines)
Ability to use human tissue/cells
Potential for use in high through-out screeningassays
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Examples of Cell-based InVitro Assays
Hepatocyte/liver slice cultures
Fibroblast/cell line cultures 3-Dimensional Cell cultures
Skin, eye irritation tests
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What is the MTT assay?
Cultured cancer cells are grown in the presence of potential
drug for a specific time period (e.g. 24,48,72, 96 h)
The amount of viable cells remaining can be determinedspectrophotometrically
Living cells convert the yellow water soluble-tetrazoliumsalt into an insoluble purple formazan crystal
Crystal is dissolved in a suitable solvent (e.g. DMSO,
acid-IPA) and the absorbance at 570 nm is obtained
A dose response curve is plotted and the concentration
at 50% cell growth is obtained
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N
N
NN
S
N
Br-
N
HN
N
N
SN
Living cancer cells
Mitocondrial dehydrogenases
MTT Formazan
MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
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Lactate dehydrogenase (LDH))
When a cell dies its LDH is released to the blood. LDH is one of
the main components in blood tests. High level of LDH areindicative of heart attacks, cancer or anemia.
If LDH levels are high, another test is performed to test the
level of the different isozymes, so that the source of theproblem can be more accurately inferred
The enzyme catalyzes the following biochemical reaction:
Pyruvate + NADH Lactate + NAD
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Real-Time PCR
mRNA quantification
DNAmRNAprotein
Reflect level of gene expression
Information about cell response
Protein production
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Real time PCR
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Real-t ime PCR App l icat ions
Viral Quantitation Quantitation of Gene Expression
Array Verification
Drug Therapy Efficacy
DNA Damage measurement
Quality Control and Assay Validation
Pathogen detection
Genotyping
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CASPASE 3
ACTIVATION ASSAY
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ANNEXIN V / PI STAINING
Annexin V binds strongly to phosphatidyl serine
which is normally excluded from the extra vascular
side of plasma membrane but flips between the inner
and outer side upon the onset of apoptosis.
Annexin V is therefore be useful to detect apoptotic
cell.
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Stress response
detection of ROS
Cellular stress response investigated with H2DCF-
DA (2-7-dichloro dihydroflouroscene diacetate).
The acetylated non-flouroscence mole.is taken up bycell culture cells,is presumbly trapped in a cytosol
by deacetylation and becomes flouroscent upon
intracellular oxidation.
A possible increase in flouoroscency of DCF.
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Comet Assay
Single Cell Gel Electrophoresis