Standard techniques for toxicology assessment

The MTT and WST-1 assays.

Cytotoxicity

Several tests are currently used in the laboratory, assessing cell metabolic activity (MTT, WST-1), cell membrane integrity (LDH), dye exclusion methods (trypan blue, propidium iodide).

  • MTT and WST-1 assays estimate cellular viability through the relative quantitation of mitochondrial enzymatic activity (succinate deshydrogenase). Cells are seeded in 96-well plates, exposed to toxicants, then MTT or WST-1 substrate is added to the exposed wells. Enzymatic activity in live cells leads to the transformation of MTT or WST-1 into purple formazan, thus enabling the detection of cellular metabolic activity through spectrophotometric reading.
  • LDH assay measures the damage to cell membrane through quantification of released lactate dehydrogenase.
  • Trypan blue assay is based on the trypan blue dye, which is internalized in cells, but immediately rejected by live cells via an ATP-dependent pathway. Dead cells (without ATP) thus remain blue and cellular viability rates can be determined by counting blue and unstained cells.
 

A comet, indicating DNA strand breaks.

Genotoxicity

  • γ-H2AX foci counting: The Histone 2 H2AX is phosphorylated in response to DNA double strand breaks (DSBs) generated by exogenous genotoxic agents. It forms a focus at the DSB location, which can be detected with immunological techniques. In particular, immunofluorescence allows the visualization of both cell nuclei and γ-H2AX foci, which indicates how many DSBs have formed in the cells.
  • Micronucleus assay: a micronucleus is a small DNA portion which comes from broken chromosomes, or chromosomes unable to migrate during cell division. Although they can be found in normal cells, they are more prone to appear in cells exposed to toxicants with clastogenic or aneugenic activity. Micronuclei are stained with DNA marking dyes (e.g. DAPI) and counted under a microscope in binucleated cells, which is achieved by culturing cells with cytokinesis-inhibitor cytochalasine B.
  • Comet assay: this assay is based on electrophoretic migration of broken DNA. Several version of the assay exist, particularly the alkaline version which makes also possible the visualization of alkali-labile sites, and the fpg and/or endoIII version which makes possible the visualizaion of oxidative DNA damage.
 

Oxidative stress

Oxidative stress occurs when a toxicant causes imbalance of pro- and anti-oxydants activities in cells. This stress is classically assessed via quantification of reactive oxygen species (ROS), of molecular antioxydants such as glutathion, or of the activity of antioxydant enzymes (catalase, superoxide dismutase, glutathion peroxydase, glutathion reductase).

  • The 2,7-Dichlorodihydrofluorescein diacetate, or H2-DCF-DA assay is a general oxidative stress assay which measures the intracellular accumulation of ROS. The H2-DCF-DA probe enters the cells, and is cleaved into DCFH by intracellular esterases. Intracellular ROS then oxidize DCFH into fluorescent DCF. Therefore global ROS content is measured by fluorescent spectroscopy or flow cytometry.
 

Molecular biology

  • RT-qPCR (Reverse-Transcription quantitative Polymerase Chain Reaction) allows the relative quantitation of genes expression levels. RNA is extracted from exposed cells and reverse transcripted into complementary DNA (cDNA - matching genomic DNA without introns). Finally cDNA levels corresponding to the genes of interest are compared through qPCR using specific DNA primers.
  • Western-blotting allows the relative quantitation of proteins expression levels. Cells are harvested, then proteins are extracted, separated through SDS-PAGE (Sodium Dodecyl Sulfate – PolyAcrylamide Gel Electrophoresis), and transferred on nitrocellulose membranes. These membranes are incubated with specific antibodies for the proteins of interest and protein expression levels are quantified using chemiluminescence.
 

Maj : 18/04/2014 (1012)

 

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