Background
The predictive value of in vitro cytotoxicity tests
is based on the idea of ‘basal’ cytotoxicity – that
toxic chemicals affect basic functions of cells which
are common to all cells, and that the toxicity can
be measured by assessing cellular damage. The
development of in vitro cytotoxicity assays has
been driven by the need to rapidly evaluate the
potential toxicity of large numbers of compounds,
to limit animal experimentation whenever possible,
and to carry out tests with small quantities of
compound. Evidence for the utility of in vitro
cytotoxicity tests1,2,3 has led many pharmaceutical
companies to screen compound libraries to remove
potentially toxic compounds early in the drug
discovery process. Early identification of toxic
effects can help project teams prioritize between
chemical series and identify Structure-Toxicity
Relationships3 to reduce cost downstream.
Assay Principle
There are 3 basic parameters upon which these
measurements are based. The first assay type is
the measurement of cellular metabolic activity. An
early indication of cellular damage is a reduction
in metabolic activity. Tests which can measure
metabolic function measure cellular ATP levels
or mitochondrial activity (via MTS metabolism).
Another parameter often tested is the measurement
of membrane integrity. The cell membrane forms a
functional barrier around the cell, and traffic into and
out of the cell is highly regulated by transporters,
receptors and secretion pathways. When cells are
damaged, they become ‘leaky’ and this forms
the basis for the second type of assay. Membrane
integrity is determined by measuring lactate
dehyrogenase (LDH) in the extracellular medium.
This enzyme is normally present in the cytosol,
and cannot be measured extracellularly unless cell
damage has occurred. Other assays measure the
uptake of fluorescent dye (ethidium DI) normally
excluded from intact cells. It has been shown that
changes in metabolic activity are better indicators
of early cell injury, and that effects on membrane
integrity are indicative of more serious injury,
leading to cell death2
. The third type of assay is
the direct measure of cell number, since dead cells
normally detach from a culture plate, and are
washed away in the medium. Cell number can
be measured by direct cell counting, or by the
measurement of total cell protein or DNA, which
are proportional to the number of cells.
Key Features of the Assay
• low compound consumption
• multiple assays: DNA, ATP, MTS, membrane
integrity
• six cell lines from different tissues available: liver,
kidney, lung, brain, testis, ovary
Assay Applications
• prioritization of hits based on cytotoxicity
• assessment of compound library for cytotoxicity