Abstract
The determination of the cytotoxic potential of new and so far unknown compounds as well as their metabolites is fundamental in risk assessment. A variety of strategic endpoints have been defined to describe toxin-cell interactions, leading to prediction of cell fate. They involve measurement of metabolic endpoints, bio-energetic parameters or morphological cell modifications. Here, we evaluated alterations of the free cytosolic Ca2+ homeostasis using the Fluo-4 dye and compared results with the metabolic cell viability assay Alamar Blue. We investigated a panel of toxins (As2O3, gossypol, H2O2, staurosporine, and titanium(IV)-salane complexes) in four different mammalian cell lines covering three different species (human, mouse, and African green monkey). All tested compounds induced an increase in free cytosolic Ca2+ within the first 5 s after toxin application. Cytosolic Ca2+ shifts occurred independently of the chemical structure in all tested cell systems and were persistent up to 3 h. The linear increase of free cytosolic Ca2+ within the first 5 s of drug treatment correlates with the EC25 and EC75 values obtained in Alamar Blue assays one day after toxin exposure. Moreover, a rise of cytosolic Ca2+ was detectable independent of induced cell death mode as assessed by caspase and poly(ADP-ribose) polymerase (PARP) activity in HeLa versus MCF-7 cells at very low concentrations. In conclusion, a cytotoxicity assay based on Ca2+ shifts has a low limit of detection (LOD), is less time consuming (at least 24 times faster) compared to the cell viability assay Alamar Blue and is suitable for high-troughput-screening (HTS).
Highlights
The development of assays estimating the cytotoxic potential of drugs and chemicals is of fundamental interest in early risk assessment to prioritize them for further testing
We show that As2O3, gossypol, H2O2 and staurosporine induce a dose-dependent increase in cytosolic Ca2+ at lethal (EC75) and sublethal (EC25) concentrations immediately after application in all tested cell lines
The Ca2+ sensitive marker Fluo-4 is bio-activated in human, murine and monkey cells Cytosolic Ca2+ was assessed using the fluorescence dye Fluo-4 (Figure 1B,C)
Summary
The development of assays estimating the cytotoxic potential of drugs and chemicals is of fundamental interest in early risk assessment to prioritize them for further testing. A few years ago, the European Union (EU) initiated a regulation for the Registration, Evaluation and Authorisation of Chemicals (REACH). Around 30 000 chemical substances, which are manufactured, imported or, used in the EU require validation [1,2]. The implementation of REACH will increase the demand of cytotoxicity testing and risk assessment. A variety of different biological endpoints have been defined for cytotoxicity testing. These include the assessment of energy status (ATP depletion, ATP/ ADP ratio), cell membrane integrity (Neutral red, Trypan blue, lactate dehydrogenase (LDH) leakage), DNA-strand
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