Introduction
Toxicity is a complex event in vivo and exhibits a wide spectrum of effects from cell death to metabolic aberrations, which result in functional changes in cells rather than death.
A. Assays based on cell viability and cell survival
Traditionally, Cytotoxicity assays have been based on indices of cell viability and survival. Viability is an instantaneous parameter thought to be predicted of survival.
Cytotoxicity assays can be divided into three groups:
1. Those that infer viability by determination of a change in membrane permeability or metabolism.
2. Those that determine viability trough absolute long-term survival, as quantified by the capacity to regenerate (Clonogenicity.)
3. Those that determine survival in an altered state by expressing a generic mutation or malignant transformation.
Viability assays
Viability assays include:
Dye exclusion methods.
Dye uptake methods.
Fluorescent methods.
Dye release methods.
Dye exclusion methods:
Viable cells exclude a number of vital dyes. A cell suspension is combined with dye and examined using a haemocytometer and optical microscope. The number of unstained cells is expressed as a percentage of the total population. Staining for viability assessment is more suited to suspensions than monolayer cultures, as dead cells detach from the monolayer.
Dye uptake methods:
These methods can be adopted for use with flow cytometers and microtiter plates. Neutral red is taken up into viable cells in culture and this property can be used in a microtiter plate dye uptake assay. The method that follows is based on that of Borenfreund and puerner.
Fluorescent methods:
Viable cells also take up Diacetyl fluorescein, which can subsequently be hydrolyzed to fluorescein, causing viable cells to fluoresce green. Non viable cells can be visualized using ethidium or propidium bromide, which causes them to fluoresce red. Viability is expressed as the number of cells that fluoresce green expressed as a percentage of total cells counted (which includes both the green and red cells counted.)
Dye release methods:
Dye release assays are exemplified by the neutral red release assay. A confluent monoloayer of cells is preloaded with dye, and the medium is then replaced with fresh dye-free medium. The cells are then exposed to a test substance for 1 minute and washed to remove liberated dye. A fixing agent is then added and optical density measurements taken from the resulting solutions in the wells of the plate. Owing to the short exposure time this test is best employed to test rapidly occurring effects directed at the cell membrane and not those compounds that require activation.
Long term survival
Often there is adelay of several hours before cells exhibit responses to toxic insult; a different type of assay is required in these cases taking into account the detachment of dead cells. These assays often measure compromised metabolic or proliferative capacity to infer long-term damage rather than short-term toxicity, which may be reversible. This can be done by metabolism and protein assays.
Metabolism assays:
Alterations in metabolism ex: changes in processes like glycolysis, enzyme activities and the ability to incorporate labeled precursors such as thymidine have all been used to measure response to toxic stimuli.
Protein content:
Protein content is a useful measure of total cellular material as an estimation of cell number, although it will vary with cell size and stage in the cell cycle. While the absorbance of protein at 280nm can be determined directly, colorimetric assays are more sensitive.
Irritancy assays
Effects on intracellular metabolism can be reflected by a decrease in extracellular acidification rate as determined by a silicon microphysiometer. Toxin membrano-lytic activity and its ability to cause protein denaturation in mammalian erythrocytes has also been used as a measure of potential ocular irritancy.
Conclusion
All the approaches have their own advantages and disadvantages. So a cluster of assays are to be performed for proper results.