Cytotoxicity

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Cytotoxicity is the quality of being toxic to cells. Examples of toxic agents are toxic metals, toxic chemicals, microbe neurotoxins, radiation particles and even specific neurotransmitters when the system is out of balance. Also some types of venom, e.g. from the puff adder (Bitis arietans) or brown recluse spider (Loxosceles reclusa) are toxic to cells.

Contents

Cell physiology

Treating cells with the cytotoxic compound can result in a variety of cell fates. The cells may undergo necrosis, 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. [1]

Measurement

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. [2]

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. [1] 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. LDH reduces NAD to NADH which elicits a colour change by interaction with a specific probe. [3] 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. [4]

Cytotoxicity can also be monitored using the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) or with 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), which yields a water-soluble product, or the 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. [1] Such ATP-based assays include bioluminescent assays in which ATP is the limiting reagent for the luciferase reaction. [5]

Cytotoxicity can also be measured by the sulforhodamine B (SRB) assay, WST assay and clonogenic assay.

Suitable assays can be combined and performed sequentially on the same cells in order to reduce assay-specific false positive or false negative results. A possible combination is LDH-XTT-NR (Neutral red assay)-SRB which is also available in a kit format.

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.

Prediction

A highly important topic is the prediction of cytotoxicity of chemical compounds based on previous measurements, i.e. in-silico testing. [6] For this purpose many QSAR and virtual screening methods have been suggested. An independent comparison of these methods has been done within the "Toxicology in the 21st century" project. [7]

Cancers

Some chemotherapies contain cytotoxic drugs, whose purpose is interfering with the cell division. These drugs cannot distinguish between normal and malignant cells, but they inhibit the overall process of cell division with the purpose to kill the cancers before the hosts. [8] [9]

Immune system

Antibody-dependent cell-mediated cytotoxicity (ADCC) describes the cell-killing ability of certain lymphocytes, 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 lymphocytes are distinguished:

See also

Related Research Articles

<span class="mw-page-title-main">Apoptosis</span> Programmed cell death in multicellular organisms

Apoptosis is a form of programmed cell death that occurs in multicellular organisms and in some eukaryotic, single-celled microorganisms such as yeast. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, and mRNA decay. The average adult human loses between 50 and 70 billion cells each day due to apoptosis. For an average human child between eight and fourteen years old, each day the approximate loss is 20 to 30 billion cells.

<span class="mw-page-title-main">Necrosis</span> Unprogrammed cell death caused by external cell injury

Necrosis is a form of cell injury which results in the premature death of cells in living tissue by autolysis. The term "necrosis" came about in the mid-19th century and is commonly attributed to German pathologist Rudolf Virchow, who is often regarded as one of the founders of modern pathology. Necrosis is caused by factors external to the cell or tissue, such as infection, or trauma which result in the unregulated digestion of cell components. In contrast, apoptosis is a naturally occurring programmed and targeted cause of cellular death. While apoptosis often provides beneficial effects to the organism, necrosis is almost always detrimental and can be fatal.

<span class="mw-page-title-main">Cytotoxic T cell</span> T cell that kills infected, damaged or cancerous cells

A cytotoxic T cell (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cell or killer T cell) is a T lymphocyte (a type of white blood cell) that kills cancer cells, cells that are infected by intracellular pathogens (such as viruses or bacteria), or cells that are damaged in other ways.

<span class="mw-page-title-main">Tumor necrosis factor</span> Protein

Tumor necrosis factor is an adipokine and a cytokine. TNF is a member of the TNF superfamily, which consists of various transmembrane proteins with a homologous TNF domain.

<span class="mw-page-title-main">Immunosuppressive drug</span> Drug that inhibits activity of immune system

Immunosuppressive drugs, also known as immunosuppressive agents, immunosuppressants and antirejection medications, are drugs that inhibit or prevent the activity of the immune system.

In the field of genetics, a suicide gene is a gene that will cause a cell to kill itself through the process of apoptosis. Activation of a suicide gene can cause death through a variety of pathways, but one important cellular "switch" to induce apoptosis is the p53 protein. Stimulation or introduction of suicide genes is a potential way of treating cancer or other proliferative diseases.

<span class="mw-page-title-main">MTT assay</span> Colorimetric analysis for measuring activity of cellular enzymes that reduce a tetrazolium dye

The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT, which is chemically 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, to its insoluble formazan, which has a purple color. Other closely related tetrazolium dyes including XTT, MTS and the WSTs, are used in conjunction with the intermediate electron acceptor, 1-methoxy phenazine methosulfate (PMS). With WST-1, which is cell-impermeable, reduction occurs outside the cell via plasma membrane electron transport. However, this traditionally assumed explanation is currently contended as proof has also been found of MTT reduction to formazan in lipidic cellular structures without apparent involvement of oxidoreductases.

Granzymes are serine proteases released by cytoplasmic granules within cytotoxic T cells and natural killer (NK) cells. They induce programmed cell death (apoptosis) in the target cell, thus eliminating cells that have become cancerous or are infected with viruses or bacteria. Granzymes also kill bacteria and inhibit viral replication. In NK cells and T cells, granzymes are packaged in cytotoxic granules along with perforin. Granzymes can also be detected in the rough endoplasmic reticulum, golgi complex, and the trans-golgi reticulum. The contents of the cytotoxic granules function to permit entry of the granzymes into the target cell cytosol. The granules are released into an immune synapse formed with a target cell, where perforin mediates the delivery of the granzymes into endosomes in the target cell, and finally into the target cell cytosol. Granzymes are part of the serine esterase family. They are closely related to other immune serine proteases expressed by innate immune cells, such as neutrophil elastase and cathepsin G.

This is a list of AIDS-related topics, many of which were originally taken from the public domain U.S. Department of Health Glossary of HIV/AIDS-Related Terms, 4th Edition.

<span class="mw-page-title-main">Fas ligand</span> Protein-coding gene in the species Homo sapiens

Fas ligand is a type-II transmembrane protein expressed on cytotoxic T lymphocytes and natural killer (NK) cells. Its binding with Fas receptor (FasR) induces programmed cell death in the FasR-carrying target cell. Fas ligand/receptor interactions play an important role in the regulation of the immune system and the progression of cancer.

<span class="mw-page-title-main">Antibody-dependent cellular cytotoxicity</span> Cell-mediated killing of other cells mediated by antibodies

Antibody-dependent cellular cytotoxicity (ADCC), also referred to as antibody-dependent cell-mediated cytotoxicity, is a mechanism of cell-mediated immune defense whereby an effector cell of the immune system kills a target cell, whose membrane-surface antigens have been bound by specific antibodies. It is one of the mechanisms through which antibodies, as part of the humoral immune response, can act to limit and contain infection.

The formazans are compounds of the general formula [R-N=N-C(R')=N-NH-R"], formally derivatives of formazan [H2NN=CHN=NH], unknown in free form.

<span class="mw-page-title-main">Fas receptor</span> Protein found in humans

The Fas receptor, also known as Fas, FasR, apoptosis antigen 1, cluster of differentiation 95 (CD95) or tumor necrosis factor receptor superfamily member 6 (TNFRSF6), is a protein that in humans is encoded by the FAS gene. Fas was first identified using a monoclonal antibody generated by immunizing mice with the FS-7 cell line. Thus, the name Fas is derived from FS-7-associated surface antigen.

<span class="mw-page-title-main">Survivin</span> Mammalian protein

Survivin, also called baculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5, is a protein that, in humans, is encoded by the BIRC5 gene.

<span class="mw-page-title-main">Trogocytosis</span>

Trogocytosis is when a cell nibbles another cell. It is a process whereby lymphocytes conjugated to antigen-presenting cells extract surface molecules from these cells and express them on their own surface. The molecular reorganization occurring at the interface between the lymphocyte and the antigen-presenting cell during conjugation is also called "immunological synapse".

<span class="mw-page-title-main">Viability assay</span> Assay created to determine survival of organs, cells, or tissues

A viability assay is an assay that is created to determine the ability of organs, cells or tissues to maintain or recover a state of survival. Viability can be distinguished from the all-or-nothing states of life and death by the use of a quantifiable index that ranges between the integers of 0 and 1 or, if more easily understood, the range of 0% and 100%. Viability can be observed through the physical properties of cells, tissues, and organs. Some of these include mechanical activity, motility, such as with spermatozoa and granulocytes, the contraction of muscle tissue or cells, mitotic activity in cellular functions, and more. Viability assays provide a more precise basis for measurement of an organism's level of vitality.

Gene expression profiling has revealed that diffuse large B-cell lymphoma (DLBCL) is composed of at least 3 different sub-groups, each having distinct oncogenic mechanisms that respond to therapies in different ways. Germinal Center B-Cell like (GCB) DLBCLs appear to arise from normal germinal center B cells, while Activated B-cell like (ABC) DLBCLs are thought to arise from postgerminal center B cells that are arrested during plasmacytic differentiation. The differences in gene expression between GCB DLBCL and ABC DLBCL are as vast as the differences between distinct types of leukemia, but these conditions have historically been grouped together and treated as the same disease.

<span class="mw-page-title-main">Paraptosis</span> Type of programmed cell death distinct from apoptosis and necrosis

Paraptosis is a type of programmed cell death, morphologically distinct from apoptosis and necrosis. The defining features of paraptosis are cytoplasmic vacuolation, independent of caspase activation and inhibition, and lack of apoptotic morphology. Paraptosis lacks several of the hallmark characteristics of apoptosis, such as membrane blebbing, chromatin condensation, and nuclear fragmentation. Like apoptosis and other types of programmed cell death, the cell is involved in causing its own death, and gene expression is required. This is in contrast to necrosis, which is non-programmed cell death that results from injury to the cell.

Complement-dependent cytotoxicity (CDC) is an effector function of IgG and IgM antibodies. When they are bound to surface antigen on target cell, the classical complement pathway is triggered by bonding protein C1q to these antibodies, resulting in formation of a membrane attack complex (MAC) and target cell lysis.

Passive antibody therapy, also called serum therapy, is a subtype of passive immunotherapy that administers antibodies to target and kill pathogens or cancer cells. It is designed to draw support from foreign antibodies that are donated from a person, extracted from animals, or made in the laboratory to elicit an immune response instead of relying on the innate immune system to fight disease. It has a long history from the 18th century for treating infectious diseases and is now a common cancer treatment. The mechanism of actions include: antagonistic and agonistic reaction, complement-dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC).

References

  1. 1 2 3 Riss TL, Moravec RA (February 2004). "Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays". Assay Drug Dev Technol. 2 (1): 51–62. doi:10.1089/154065804322966315. PMID   15090210.
  2. ↑ Gavanji S, Bakhtari A, Famurewa AC, Othman EM (January 2023). "Cytotoxic Activity of Herbal Medicines as Assessed in Vitro: A Review". Chemistry & Biodiversity. 20 (2): 3–27. doi: 10.1002/cbdv.202201098 . PMID   36595710. S2CID   255473013.
  3. ↑ Decker T, Lohmann-Matthes ML (November 1988). "A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity". J. Immunol. Methods. 115 (1): 61–9. doi:10.1016/0022-1759(88)90310-9. PMID   3192948.
  4. ↑ Niles AL, Moravec RA, Eric Hesselberth P, Scurria MA, Daily WJ, Riss TL (July 2007). "A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers". Anal. Biochem. 366 (2): 197–206. doi:10.1016/j.ab.2007.04.007. PMID   17512890.
  5. ↑ Fan F, Wood KV (February 2007). "Bioluminescent assays for high-throughput screening". Assay Drug Dev Technol. 5 (1): 127–36. doi:10.1089/adt.2006.053. PMID   17355205. S2CID   10261888.
  6. ↑ Dearden, J. C. (2003). "In silico prediction of drug toxicity". Journal of Computer-aided Molecular Design. 17 (2–4): 119–127. Bibcode:2003JCAMD..17..119D. doi:10.1023/A:1025361621494. PMID   13677480. S2CID   21518449.
  7. ↑ "Toxicology in the 21st century Data Challenge" https://tripod.nih.gov/tox21/challenge/leaderboard.jsp
  8. ↑ Priestman, T. J. (1989). Cancer Chemotherapy: an Introduction. doi:10.1007/978-1-4471-1686-8. ISBN   978-3-540-19551-1. S2CID   20058092.
  9. ↑ "How Is Chemotherapy Used to Treat Cancer?". www.cancer.org. Retrieved 2021-06-28.
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