Cytotoxicity
Encyclopedia
Cytotoxicity is the quality of being toxic
to cell
s. Examples of toxic agents are a chemical substance
, an immune cell or some types of venom
(e.g. from the puff adder
or brown recluse spider
).
, in which they lose membrane integrity and die rapidly as a result of cell lysis. The cells can stop actively growing and dividing (a decrease in cell viability), or the cells can activate a genetic program of controlled cell death (apoptosis
).
Cells undergoing necrosis typically exhibit rapid swelling, lose membrane integrity, shut down metabolism and release their contents into the environment. Cells that undergo rapid necrosis in vitro do not have sufficient time or energy to activate apoptotic machinery and will not express apoptotic markers. Apoptosis is characterized by well defined cytological and molecular events including a change in the refractive index
of the cell, cytoplasmic shrinkage, nuclear condensation and cleavage of DNA into regularly sized fragments. Cells in culture that are undergoing apoptosis eventually undergo secondary necrosis. They will shut down metabolism, lose membrane integrity and lyse.
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 commonly measured molecule is lactate dehydrogenase
(LDH). 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 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.
s, which requires the target cell being marked by an antibody
. Lymphocyte-mediated cytotoxicity, on the other hand, does not have to be mediated by antibodies; nor does complement-dependent cytotoxicity (CDC), which is mediated by the complement system
.
Three groups of cytotoxic lymphocyte
s are distinguished:
Toxicity
Toxicity is the degree to which a substance can damage a living or non-living organisms. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a substructure of the organism, such as a cell or an organ , such as the liver...
to cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
s. Examples of toxic agents are a chemical substance
Chemical substance
In chemistry, a chemical substance is a form of matter that has constant chemical composition and characteristic properties. It cannot be separated into components by physical separation methods, i.e. without breaking chemical bonds. They can be solids, liquids or gases.Chemical substances are...
, an immune cell or some types of venom
Venom
Venom is the general term referring to any variety of toxins used by certain types of animals that inject it into their victims by the means of a bite or a sting...
(e.g. from the puff adder
Bitis arietans
Bitis arietans is a venomous viper species found in savannah and grasslands from Morocco and western Arabia throughout Africa except for the Sahara and rain forest regions. It is responsible for causing the most fatalities in Africa owing to various factors, such as its wide distribution and...
or brown recluse spider
Brown recluse spider
The brown recluse spider or violin spider, Loxosceles reclusa, is a member of the family Sicariidae . The spider has a venomous bite....
).
Cell physiology
Treating cells with a cytotoxic compound can result in a variety of cell fates. The cells may undergo necrosisNecrosis
Necrosis is the premature death of cells in living tissue. Necrosis is caused by factors external to the cell or tissue, such as infection, toxins, or trauma. This is in contrast to apoptosis, which is a naturally occurring cause of cellular death...
, in which they lose membrane integrity and die rapidly as a result of cell lysis. The cells can stop actively growing and dividing (a decrease in cell viability), or the cells can activate a genetic program of controlled cell death (apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
).
Cells undergoing necrosis typically exhibit rapid swelling, lose membrane integrity, shut down metabolism and release their contents into the environment. Cells that undergo rapid necrosis in vitro do not have sufficient time or energy to activate apoptotic machinery and will not express apoptotic markers. Apoptosis is characterized by well defined cytological and molecular events including a change in the refractive index
Refractive index
In optics the refractive index or index of refraction of a substance or medium is a measure of the speed of light in that medium. It is expressed as a ratio of the speed of light in vacuum relative to that in the considered medium....
of the cell, cytoplasmic shrinkage, nuclear condensation and cleavage of DNA into regularly sized fragments. Cells in culture that are undergoing apoptosis eventually undergo secondary necrosis. They will shut down metabolism, lose membrane integrity and lyse.
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
Trypan blue
Trypan blue is a vital stain used to selectively colour dead tissues or cells blue. It is a diazo dye.Live cells or tissues with intact cell membranes are not coloured. Since cells are very selective in the compounds that pass through the membrane, in a viable cell trypan blue is not absorbed;...
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 commonly measured molecule is lactate dehydrogenase
Lactate dehydrogenase
Lactate dehydrogenase is an enzyme present in a wide variety of organisms, including plants and animals.Lactate dehydrogenases exist in four distinct enzyme classes. Two of them are cytochrome c-dependent enzymes, each acting on either D-lactate or L-lactate...
(LDH). 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 MTT
MTT assay
The MTT assay and the MTS assay are colorimetric assays for measuring the activity of enzymes that reduce MTT or close dyes to formazan dyes, giving a purple color. A main application allows to assess the viability and the proliferation of cells...
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
Formazan
Formazan dyes are artificial chromogenic products of the reduction of tetrazolium salts by dehydrogenases and reductases. They have a variety of colors from dark blue to deep red to orange, depending on the original tetrazolium salt used as the substrate for the reaction.Leading examples of...
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
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
content as a marker of viability. Such ATP-based assays include bioluminescent assays in which ATP is the limiting reagent for the luciferase
Luciferase
Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence and is distinct from a photoprotein. One famous example is the firefly luciferase from the firefly Photinus pyralis. "Firefly luciferase" as a laboratory reagent usually refers to P...
reaction.
Cytotoxicity can also be measured by the sulforhodamine B
Sulforhodamine b
Sulforhodamine B or kiton red is a fluorescent dye with uses spanning from laser-induced fluorescence to the quantification of cellular proteins of cultured cells. The red, solid, water-soluble dye is primarily used as a polar tracer.The dye has maximal absorbance at 565 nm light and...
(SRB) assay, WST assay and clonogenic assay
Clonogenic assay
A clonogenic assay is a microbiology technique for studying the effectiveness of specific agents on the survival and proliferation of cells. It is frequently used in cancer research laboratories to determine the effect of drugs or radiation on proliferating tumor cells as well as for titration of...
.
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
Electric cell-substrate impedance sensing
Electric cell-substrate impedance sensing or ECISTM refers to a non-invasive biophysical approach to monitor living animal cells in vitro, i.e...
(ECIS). Label-free real-time techniques provide the kinetics of the cytotoxic response rather than just a snapshot like many colorimetric endpoint assays.
Immune system cytotoxicity
Antibody-dependent cell-mediated cytotoxicity (ADCC) describes the cell-killing ability of certain lymphocyteLymphocyte
A lymphocyte is a type of white blood cell in the vertebrate immune system.Under the microscope, lymphocytes can be divided into large lymphocytes and small lymphocytes. Large granular lymphocytes include natural killer cells...
s, which requires the target cell being marked by an antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
. Lymphocyte-mediated cytotoxicity, on the other hand, does not have to be mediated by antibodies; nor does complement-dependent cytotoxicity (CDC), which is mediated by the complement system
Complement system
The complement system helps or “complements” the ability of antibodies and phagocytic cells to clear pathogens from an organism. It is part of the immune system called the innate immune system that is not adaptable and does not change over the course of an individual's lifetime...
.
Three groups of cytotoxic lymphocyte
Lymphocyte
A lymphocyte is a type of white blood cell in the vertebrate immune system.Under the microscope, lymphocytes can be divided into large lymphocytes and small lymphocytes. Large granular lymphocytes include natural killer cells...
s are distinguished:
- Cytotoxic T cellCytotoxic T cellA cytotoxic T cell belongs to a sub-group of T lymphocytes that are capable of inducing the death of infected somatic or tumor cells; they kill cells that are infected with viruses , or are otherwise damaged or...
s - Natural killer cellNatural killer cellNatural killer cells are a type of cytotoxic lymphocyte that constitute a major component of the innate immune system. NK cells play a major role in the rejection of tumors and cells infected by viruses...
s - Natural killer T cellNatural Killer T cellNatural killer T cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. Many of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds self- and foreign lipids and glycolipids...
s