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Cytotoxicity Tests For High-Throughput Drug Discovery
Cytotoxicity Tests For High-Throughput Drug Discovery
Cytotoxicity Tests For High-Throughput Drug Discovery
Introduction
This review will discuss the challenges facing the pharmaceutical industry resulting from the vast increases in
sample numbers produced by high-throughput screening
(HTS). Specifically, it will focus on the bottle necks created by increased demand for cytotoxicity testing (required
to assess compound safety) and will address the rapid
methods that are being developed to overcome this growing problem. Much of the information was obtained from
the Society for Biomolecular Screening (SBS) annual conference in Vancouver (69 September 2000). Otherwise
the reviewed articles cover the year between October 1999
and October 2000. Occasionally, I have made reference to
older papers in order to illustrate a point where necessary.
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within a population may proliferate following drug treatment. He therefore stresses that it is the absolute number
of surviving cells that is the most important parameter
rather than the percentage of dead cells, which is often
determined by flow cytometry. This is an observation that
we have regularly noticed in our own laboratory.
Despite recent developments with high-speed machines [8],
flow cytometry is currently not suited to the demands of
high-throughput cytotoxicity testing. High-throughput cytotoxicity assays need to be performed in microplates where
incubation and analysis can be achieved in the same plate and
a number of fluorescent microplate assays have been developed. Wodnicka et al. [9] have used a ruthenium dye (tris
4,7-diphenyl-1, 10-phenanthroline ruthenium [II] chloride) to
act as a biosensor. The fluorescence produced by this dye is
substantially quenched in the presence of oxygen. Thus, as
cells grow, the oxygen present in the medium diminishes over
time. This can be detected by an increase in fluorescence
resulting from a reduction in the quenching effect. Since the
ruthenium was not toxic to the cell types tested (CHO,
CaCo2, Alma16, MDCK, HL60, U-937, SF9, Saccharomyces
cerevisiae and Escherichia coli), it was possible to monitor the
growth of cells during the culture period by measuring
increase in fluorescence with a microplate fluorimeter.
Addition of cytotoxic agents to the cultures reversed this
effect, thus providing a very simple and rapid microplate cytotoxicity assay. From the data presented, however, it appears
that sensitivity is rather low, requiring 50,000 HL60 cells to
achieve a significant signal. Cells with higher metabolic rates,
and therefore greater oxygen consumption, could be detected with greater sensitivity and the insect cell SF9 was cited as
an example of this. Although the dynamic range of the assay
appears rather narrow (sixfold compared to a number of logs
for other assays), the method does facilitate kinetic analysis of
toxic effects, which the authors suggest may provide predictive value to indicate a drugs mode of action.
Another fluorescent dye, Alamar Blue (resazurin), lends itself
well to the demands of high-throughput cytotoxicity testing.
Oxidized, blue non-fluorescent Alamar Blue is reduced to a
pink fluorescent dye in cell-culture medium as a result of cell
activity. The exact mechanism of this dye reduction remains
to be elucidated; however, it is thought to be induced by
mitchondrial enzymes [10]. The test is very simple to perform, requiring the addition of only one reagent to the cell
culture supernatant (therefore providing a homogeneous
assay). It does not kill the cells, thus facilitating additional
tests on the same sample (which can be very important when
valuable primary cells are used). Most importantly, Alamar
Blue can provide a high degree of sensitivity, detecting a few
as 80 cells [11]. Although Alamar Blue can be co-cultured
with the cells giving the opportunity for kinetic analysis of
cytotoxicity, OBrien et al. [11] noted that compounds such as
nickel when added to cultures resulted in the reduction of the
dye even though the cells were dead, thus overestimating cell
survival. He concluded that kinetic experiments may be
unreliable unless strictly controlled.
Bioluminescent assays
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Conclusions
The objective of high-throughput screening is to reduce
the number of lead compounds to those that show true
potential of becoming genuine product candidates. As
pharmaceutical companies strive to improve the cost effectiveness of their drug-discovery programmes, it is
becoming clear that a considerable amount of money is lost
on compounds that fail late in the process because of their
toxicity. Their response to this has been to move toxicity
testing to an early phase in drug development. This is placing new demands on cytotoxicity tests, requiring
miniaturisation to 384-well formats, homogeneous mix
and measure assays and improvements in sensitivity.
Many of the established cytotoxicity endpoints, such as
tetrazolium dyes and Alamar Blue, have been successfully
miniaturised and can provide high-throughput analysis;
however, these methods were subsequently found to be
wanting because of poor sensitivity. Bioluminescent analysis of ATP appears to offer the answer to the demands of
speed and simplicity, and provides the sensitivity needed
to screen out low-level toxicity.
of special interest
of outstanding interest
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7.
King M: Detection of dead cells and measurement of cell killing by
flow cytometry. J Immunol Methods 2000, 243:155-166.
This paper provides a comprehensive discussion of the benefits and pitfalls
of a wide variety of fluorescent dyes used in cell viability testing.
8.
9.