An Overview of Cytotoxicity Assay During Drug Developmentby Maria B. Medical Expert
Cytotoxicity, as the name suggests, is the toxicity of the cells. When the cells are treated with cytotoxic compounds, it may lead to cell necrosis - leading to loss of membrane integrity, and the result is rapid death caused by cell lysis. These cells can stop growing and dividing, or the cells can activate a genetic program leading to controlled death.
The absorption of harmful chemicals causes cytotoxicity – the chemicals can kill cancer cells but have the potential to destroy the healthy cells also. In drug development, studying cytotoxicity is necessary because cytotoxic drugs are necessary for treating critical diseases. The drugs contain chemicals which are toxic to cells, preventing their replication or growth and are effective to control diseases such as cancer.
The basis of a Cytotoxicity assay assesses the damage to cellular membranes. It follows four types of methods:
- Enzyme Leakage assays
- Membrane impermeable dyes
- Amine-reactive dyes (used in live: dead cell assays)
- Dye combination live: dead cell assays
Enzyme Leakage- These assays measure the activity of enzymes that leak into the extracellular level causing cell membrane damage. The most popular assay is for lactate dehydrogenase.
Membrane Impermeable Dyes- Cell viability assays often use membrane-impermeable fluorescent dyes (mostly DNA stains) that stain cells with damaged cell membranes. DRAQ7 and 7-AAD have largely replaced propidium iodide for cell viability assays due to its broad emission spectra and tendency to bind to live cells.
Amine-reactive dyes for Live: Dead Cell Assays- Amine-reactive dyes weakly stain viable cells by binding to cell surface amines and strongly stain membrane-compromised cells by reacting with intracellular amines. The fluorescence level can differentiate dead and live cells.
Combined Dye Live: Dead Cell Assays- Multiple dyes can be combined in a single live:dead cell assay. Examples include the popular live and dead cells assays ab115347 with ethidium homodimer to label dead cells and an esterase-cleaved dye for live cells. The alternative, cell viability assay kit (fluorometric-dual green/red) ab112121, includes a red DNA staining dye for dead cells and a green esterase-cleaved dye for live cells.
Advantages of Cytotoxicity Assays
They are a quick way to assess the effects of a chemical compound on a given human cell line. The most well-known techniques are the MTT-and the LD- assays. These tests are cheap and easy to execute but are not very precise and dependant on various environmental factors. They do not the time-dependency of the toxic effect.
The importance of Cytotoxicity assays in early drug development is unquestionable even though no assay technology for detecting cytotoxicity in vitro is perfect. Based on membrane integrity changes, positive-readout assay is typically indicated for shorter-term exposure models. These assays may not correctly determine the absolute degree of early or late-stage cytotoxicity since the kinetics of biomarker emergence or degradation. In early drug discovery and development, an in vitro evaluation of new drug candidates is tested for reliability and in vivo correlatability. It can be concluded that it is important to put in vitro toxicity data into consideration during pharmaceutical development.
Created on Jul 17th 2019 02:38. Viewed 509 times.