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.2013 Feb 6;11(1):11.
doi: 10.1186/1478-811X-11-11.

Cytosolic Ca2+ shifts as early markers of cytotoxicity

Affiliations

Cytosolic Ca2+ shifts as early markers of cytotoxicity

Philippe Wyrsch et al. Cell Commun Signal..

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).

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Figures

Figure 1
Figure 1
Principle and experimental setup for cytotoxicity determination. (A) Alamar Blue conversion (B) Principle of Fluo-4 assay for free cytosolic Ca2+ determination (C) Experimental time line for Fluo-4 and Alamar blue assays as investigated in this study.
Figure 2
Figure 2
Toxins, cells and Fluo-4 activation capacities. (A) Chemical structures of the investigated compounds (B) List of mammalian cells analyzed in this study (C) HeLa, MCF-7, murine fibroblasts and Vero 76 cells were incubated with Fluo-4, AM as described in Methods and relative fluorescent (RFU) was determined (mean±SD; n=8).
Figure 3
Figure 3
Assessment of As2O3, gossypol, H2O2and staurosporine-induced toxicity in HeLa cells. (A) Upper panel: Alamar Blue assay in presence of As2O3 as indicated (mean±SD; n≥3; n.s. not significant;t test). Lower panel: Fluo-4 analysis of 5 μM and 50 μM As2O3 treated cells (mean±SD; *p<0.025; n=3;t test) (B) Upper panel: Alamar Blue assay in presence of gossypol as indicated (mean±SD; n≥3). Lower panel: Fluo-4 analysis of 75 μm and 100 μM gossypol treated cells (mean±SD; *p<0.0025; n≥4;t test) (C) Upper panel: Alamar Blue assay in presence of H2O2 as indicated (mean±SD; n≥4). Lower panel: Fluo-4 analysis of 0.5 mM and 2 mM H2O2 treated cells (mean±SD; *p<0.025; n≥3;t test) (D) Upper panel: Alamar Blue assay in presence of staurosporine as indicated (mean±SD; n≥3). Lower panel: Fluo-4 analysis of 400 nM and 1000 nM staurosporine treated cells (mean±SD; *p<0.05; n=3;t test).
Figure 4
Figure 4
Assessment of As2O3, gossypol, H2O2and staurosporine-induced toxicity in MCF-7 cells. (A) Upper panel: Alamar Blue assay in presence of As2O3 as indicated (mean±SD; n≥4). Lower panel: Fluo-4 analysis of 20 μM and 50 μM As2O3 treated cells (mean±SD; *p<0.05; n=3;t test) (B) Upper panel: Alamar Blue assay in presence of gossypol as indicated (mean±SD; n≥4; n.s. not significant;t test). Lower panel: Fluo-4 analysis of 60 μm and 75 μM gossypol treated cells (mean±SD; *p<0.025; n=4;t test) (C) Upper panel: Alamar Blue assay in presence of H2O2 as indicated (mean±SD; n≥4). Lower panel: Fluo-4 analysis of 5 mM and 10 mM H2O2 treated cells (mean±SD; *p<0.005; n≥3;t test) (D) Upper panel: Alamar Blue assay in presence of staurosporine as indicated (mean±SD; n≥4). Lower panel: Fluo-4 analysis of 200 nM and 400 nM staurosporine treated cells (mean±SD; *p<0.05; n≥3;t test).
Figure 5
Figure 5
Assessment of As2O3, gossypol, H2O2and staurosporine-induced toxicity in murine fibroblasts. (A) Upper panel: Cells were treated with increasing concentrations of As2O3 as indicated and afterwards cell viability was analysed with Alamar Blue (mean±SD; n≥4). Lower panel: Fluo-4 analysis of 45 μM and 50 μM As2O3 treated cells (mean±SD; *p<0.0001; n≥4;t test) (B) Upper panel: Cells were treated with increasing concentrations of gossypol as indicated and afterwards cell viability was analysed with Alamar Blue (mean±SD; n≥4). Lower panel: Fluo-4 analysis of 75 μM and 100 μM gossypol treated cells (mean±SD; *p<0.05; n=4;t test) (C) Upper panel: Alamar Blue assay in presence of H2O2 as indicated (mean±SD; n≥3). Lower panel: Fluo-4 analysis of 0.5 mM and 5 mM H2O2 treated cells (mean±SD; *p<0.01; n≥3;t test) (D) Upper panel: Alamar Blue assay in presence of staurosporine as indicated (mean±SD; n≥3). Lower panel: Fluo-4 analysis of 500 nM and 4000 nM staurosporine treated cells (mean±SD; n. s. not significant; n≥3;t test).
Figure 6
Figure 6
Assessment of As2O3, gossypol, H2O2and staurosporine-induced toxicity in Vero 76 cells. (A) Upper panel: Alamar Blue assay in presence of As2O3 as indicated (mean±SD; n≥3). Lower panel: Fluo-4 analysis of 35 μM and 100 μM As2O3 treated cells (mean±SD; *p<0.025; n=3;t test). (B) Upper panel: Alamar Blue assay in presence of gossypol as indicated (mean±SD; n≥3; n.s. not significant;t test). Lower panel: Fluo-4 analysis of 75 μm and 150 μM gossypol treated cells (mean±SD; *p<0.025; n=4;t test) (C) Upper panel: Alamar Blue assay in presence of H2O2 as indicated (mean±SD; n≥3). Lower panel: Fluo-4 analysis of 8.5 mM and 10 mM H2O2 treated cells (mean±SD; *p<0.025; n≥3;t test) (D) Upper panel: Alamar Blue assay in presence of staurosporine as indicated (mean±SD; n≥4). Lower panel: Fluo-4 analysis of 200 nM and 500 nM staurosporine treated cells (mean±SD; *p<0.0025; n=3;t test).
Figure 7
Figure 7
Caspase activation after sublethal and lethal doses of As2O3, gossypol, H2O2and staurosporine in HeLa and MCF-7 cells. (A) Western blot analyses of cleaved caspase 7 and 9 after staurosporine and As2O3 treatment in HeLa cells 4 h post treatment. Α-tubulin is shown as loading control. (B) Western blot analyses of cleaved caspase 7 and 9 after staurosporine and As2O3 treatment in MCF-7 cells 4 h post treatment. Α-tubulin and 1 μM staurosporine treated HeLa cells are shown as controls. (C) Western blot analyses of cleaved caspase 7 and 9 after gossypol treatment as indicated in HeLa and MCF-7 cells 4 h post treatment with α-tubulin as loading control. (D) Western blot analyses of cleaved caspase 7 and 9 after H2O2 treatment as indicated in HeLa and MCF-7 cells 4 h post treatment with α-tubulin as loading control. (E) Cell viability was assessed with Alamar Blue in HeLa cells in presence or absence of Q-VD-OPh (20 μM). Cells were challenged with EC75 values of As2O3, gossypol, H2O2 and staurosporine as indicated. Differences in cell survival of Q-VD-OPh plus toxin compared to toxin only treatment are shown (mean±SD; *p<0.05; n=7;t test).
Figure 8
Figure 8
PAR formation and its effect on cell survival after lethal doses of As2O3, gossypol, H2O2and staurosporine in HeLa and MCF-7 cells. (A) PAR detection by immunofluorescence in HeLa cells treated with 50 μM As2O3, 100 μM gossypol, 2 mM H2O2 or 1 μM staurosporine as described in Methods. Nuclear DAPI staining is shown as control. (B) PAR detection by immunofluorescence in MCF-7 cells treated with 50 μM As2O3, 75 μM gossypol, 10 mM H2O2 or 0.4 μM staurosporine as described with nuclear DAPI staining as control. (C) Cell viability was assessed with Alamar Blue in HeLa cells in presence or absence of PJ-34 (5 μM). Cells were challenged with EC75 values of As2O3, gossypol, H2O2 and staurosporine as indicated. Differences of cell survival are shown (mean±SD; *p<0.05; n=8;t test). (D) Cell viability was assessed with Alamar Blue in MCF-7 cells in presence or absence of PJ-34 (5 μM). Cells were challenged with EC75 values of As2O3, gossypol, H2O2 and staurosporine as indicated. Differences of cell survival are shown (mean±SD; *p<0.05; n=8;t test).
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