Here we show the results of comparing cell viability, cytotoxicity, and apoptosis assays for measuring the time- and dose-dependent toxic effects of tamoxifen on HepG2 cells. The quantitation of adenosine 5'-triphosphate (ATP), 5-(3-carboxymethoxyphenyl)-2-(4,5- dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium, inner salt (MTS) tetrazolium reduction, and resazurin reduction methods used to estimate the number of viable cells all showed a similar trend of decreased cell viability after longer periods of tamoxifen exposure to HepG2 cells. The release of lactate dehydrogenase (LDH) as a marker for cells with a compromised membrane and the increase in caspase-3/7 activity as a marker for apoptosis were both shown to increase using the same tamoxifen exposure conditions that caused a decrease in HepG2 cell viability. The longer the duration of exposure of tamoxifen, the lower the concentration required to kill or induce apoptosis in HepG2 cells. In contrast, there was no change in LDH release from HL-60 cells using conditions of vinblastine treatment that caused an increase in caspase activity and a decrease in ATP content, suggesting a difference in the mechanism of cell death between the two model systems. Both the density of parent stock cultures used as a source of cells to prepare assay plates and the density of cells per well in the assay plates were demonstrated to be factors than can influence the apparent potency of a toxin in viability, toxicity, and apoptosis assays. These results illustrate the importance of understanding the kinetics and mechanism of cell death of each in vitro model system as prerequisites for choosing the most appropriate assay method.

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Each of these methods has its relative merits and detractions; however multi-parametric methods using both viability and cytotoxicity markers may mitigate the inherent shortcomings of single parameter measures.

In vitro cytotoxicity testing has become an integral aspect of drug discovery because it is a convenient, costeffective, and predictive means of characterizing the toxic potential of new chemical entities. The early and routine implementation of this testing is testament to its prognostic importance for humans. Although a plethora of assay chemistries and methods exist for 96-well formats, few are practical and sufficiently sensitive enough for application in high throughput screening (HTS). Here we briefly describe a handful of the currently most robust and validated HTS assays for accurate and efficient assessment of cytotoxic risk. We also provide guidance for successful HTS implementation and discuss unique merits and detractions inherent in each method. Lastly, we discuss the advantages of combining specific HTS compatible assays into multi-parametric, same-well formats.

Testing the effects of compounds on the viability of cells grown in culture is widely used as a predictor of potential toxic effects in whole animals. Among the several alternative assays available, measuring the levels of ATP is the most sensitive, reliable, and convenient method for monitoring active cell metabolism. However, recently developed combinations of methods have made it possible to collect more information from in vitro cytotoxicity assays using standard fluorescence and luminescence plate readers. This chapter describes two assay methods. The first utilizes beetle luciferase for measuring the levels of ATP as a marker of viable cells. The second more recently developed multiplex method relies on selective measurement of three different protease activities as markers for viable, necrotic, and apoptotic cells. Data analysis from the measurement of three marker protease activities from the same sample provides a useful tool to help uncover the mechanism of cell death and can serve as an internal control to help identify assay artifacts.