in vitro assessment of nano material toxicity

8/12/2019 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

in vitro assessment of nano material toxicity

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