9 March 2001
Measuring Cell Death by Cytotoxity Detection Methods
Cell necrobiology (the study of mechanisms of cell death) is becoming an increasingly popular and important field in science. There are two ways in which cells can die, either passively (necrosis) or actively (apoptosis), which involves signal transduction cascades. Necrosis is accompanied by mitochondrial swelling and plasma membrane permeation, while apoptosis involves an articulated breakdown of the cell into membrane-bound particles, termed apoptotic bodies. Sometimes cytotoxic effects and processes are typically apoptotic, for instance the selection of T-lymphocytes and the effects of natural killer (NK) cells on target cells. Cell injury or death without clear morphological changes is atypical.

Pharmaceutical companies need to test the efficacy and safety of drugs and therefore must deal with complex cause-effect relationships while screening for cytotoxicity. The complexity highlights the need for "crossover" studies that involve cytotoxicity, genotoxicity and pharmacokinetics. There are a number of global screening techniques available that cover most mechanisms of cytotoxicity and cell death. Measuring altered cell permeability, discussed below, is just one of these.

Cytotoxicity assays are used to quantify cell proliferation in response to growth factors, cytokines, mitogens, or nutrients, and to analyse cytotoxic compounds such as anticancer drugs. Most of these assays measure cell viability by plasma membrane permeability. Traditionally the release of radio-isotopic labels (3H or 51Cr) was measured, but these costly and hazardous manipulations are becoming less popular with scientists. Trypan blue exclusion has also been widely used, but the technique is unsuitable for large numbers of samples, and involves meticulous manual cell counting.

Newer permeability-based cytotoxicity methods measure the release of cytoplasmic enzymes. While glutamate-dependent transaminase assays are cumbersome and the enzymes themselves not very abundant, lactate dehydrogenase (LDH)-based assays have proven useful for measuring both cell-mediated and antibody-dependent cellular cytotoxicity. LDH release has been shown to correlate very well with 51Cr release and trypan blue staining.

Another family of assays focuses on biological functions inside intact cells as an alternative to permeability testing. Viable cells reduce tetrazolium salts to coloured formazan compounds, mainly using extramitochondrial dehydrogenases.

There are other laboratory techniques for measuring cytotoxicity, including:

· Cytotoxicity assays that rely on the ability of cells to make ATP
· The comet, or single-cell gel electrophoresis (SCGE), assay - designed for genotoxicity screening, but considered one of the most interesting methods for comprehensive cytotoxicity studies
· Assays that focus on tissue or cell-type specific cytotoxicity studies

These techniques are discussed in the Profile section of The Scientist. Suppliers of the assays are also provided on this page.