Tuesday, February 3, 2015

Cytotoxicity Testing



DEFENITION

Cytotoxicity is a term used for substances to describe how toxic or poisonous to the cells they can potentially be. Exposure to cytotoxic substances can result in permanent cellular damage or even death. To determine levels of cytotoxicity, laboratory tests and assays are often conducted on substances or ingredients that will be included in any medication or medical apparatus. As for its etymology, the term “cytotoxicity” is a combination of two Greek words: “kytos,” which refers to the cell, and “toxikon,” which pertains to poison.
Substances that can be described as cytotoxic can include some chemicals or even other types of cells. When it comes to chemicals, some naturally produced ones can come in the form of animal venom, such as in some spiders and snakes. The family of vipers, for example, is known to release a type of cytotoxin called the haemotoxin, which can rupture red blood cells and cause internal bleeding and organ damage. Another dangerous cytotoxin is the cardiotoxin, which is often associated with a king cobra’s venomous bite. The toxin attaches itself to the muscle cells in the heart, causing the organ to stop pumping blood, which can result in death.

MEASURING CYTOTOXICITY

Cytotoxicity assays are widely used by the pharmaceutical industry to screen for cytotoxicity in compound libraries. Researchers can either look for cytotoxic compounds, if they are interested in developing a therapeutic that targets rapidly dividing cancer cells, for instance; or they can screen "hits" from initial high-throughput drug screens for unwanted cytotoxic effects before investing in their development as a pharmaceutical.
Assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. Compounds that have cytotoxic effects often compromise cell membrane integrity. Vital dyes, such as trypan blue or propidium iodide are normally excluded from the inside of healthy cells; however, if the cell membrane has been compromised, they freely cross the membrane and stain intracellular components.Alternatively, membrane integrity can be assessed by monitoring the passage of substances that are normally sequestered inside cells to the outside. One molecule, lactate dehydrogenase (LDH), is commonly measured using LDH assay. Protease biomarkers have been identified that allow researchers to measure relative numbers of live and dead cells within the same cell population. The live-cell protease is only active in cells that have a healthy cell membrane, and loses activity once the cell is compromised and the protease is exposed to the external environment. The dead-cell protease cannot cross the cell membrane, and can only be measured in culture media after cells have lost their membrane integrity.
Cytotoxicity can also be monitored using the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) or MTS assay. This assay measures the reducing potential of the cell using a colorimetric reaction. Viable cells will reduce the MTS reagent to a colored formazan product. A similar redox-based assay has also been developed using the fluorescent dye, resazurin. In addition to using dyes to indicate the redox potential of cells in order to monitor their viability, researchers have developed assays that use ATP content as a marker of viability. Such ATP-based assays include bioluminescent assays in which ATP is the limiting reagent for the luciferase reaction.
Cytotoxicity can also be measured by the sulforhodamine B (SRB) assay, WST assay and clonogenic assay.
A label-free approach to follow the cytotoxic response of adherent animal cells in real-time is based on electric impedance measurements when the cells are grown on gold-film electrodes. This technology is referred to as electric cell-substrate impedance sensing (ECIS). Label-free real-time techniques provide the kinetics of the cytotoxic response rather than just a snapshot like many colorimetric endpoint assays.

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