Rapid Report pubs.acs.org/crt
Copper Ions Interfere with the Reduction of the Water-Soluble Tetrazolium Salt‑8 Annetta Semisch and Andrea Hartwig* Karlsruhe Institute of Technology (KIT), Institute of Applied Biosciences, Department of Food Chemistry and Toxicology, Adenauerring 20a, 76131 Karlsruhe, Germany S Supporting Information *
ABSTRACT: Metabolic activity as a measure of cell viability is frequently determined using the water-soluble tetrazolium salt 2-(2-methoxy-4-nitrophenyl)-3(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8), commercially available as CCK-8 reagent. In this study, CCK-8 was investigated with respect to its suitability for investigating nano- and microscale copper oxide (CuO NP and CuO MP) as well as water-soluble copper chloride (CuCl2). The results were compared to cell number and colony forming ability. Our data demonstrate that the CCK-8 assay overestimates the loss of metabolic activity by CuCl2 and CuO NP, because of interference by copper ions with the reduction of the dye.
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were incubated for 1 h before the absorbance was measured (see the detailed protocol in the Supporting Information). DMEM/FCS, CuO MP, and CuCl2 suspensions exerted an intrinsic absorbance of 0.16 at 450 nm (data not shown). In the case of CuO NP suspensions, the absorbance increased in a concentration-dependent manner from 0.16 to 0.26 (Figure 1), probably because of the black color and finely dispersed state of the particles.
etabolic activity as a parameter for cell viability is commonly applied in toxicity testing and frequently determined using the commercially available reagent Cell Counting Kit-8 [CCK-8 (see the Supporting Information)]. CCK-8 contains the tetrazolium salt WST-8 and the intermediate electron carrier 1-methoxy-5-methylphenazinium methyl sulfate (mPMS).1 Adjacent to metabolically active cells, the pink WST-8 is reduced extracellularly to a yellow reaction product called formazan. Its formation can be quantified photometrically, with the amount of formazan generated being directly proportional to the metabolic activity and thus to the number of living cells of a given cell population.2 Nevertheless, several studies linked tetrazolium-based measurements to interference with metals and nanomaterials.3−8 However, to the best of our knowledge, so far no study has addressed the impact of copper compounds on the reliability of the CCK-8 assay and compared it with less error-prone methods to determine cytotoxicity like colony forming ability (CFA) and/ or cell number (CN).9 Thus, the aim of this study was to assess the suitability of the CCK-8 assay to quantify the impact of water-soluble CuCl2 as well as CuO NP and CuO MP on metabolic activity. First, the potential interference of CuO NP, CuO MP (for particle characteristics, see the Supporting Information), and CuCl2 with the detection wavelength of 450 nm was investigated; 100 μL of cell culture medium supplemented with 10% fetal calf serum (DMEM/FCS) alone or with increasing concentrations of CuO NP, CuO MP (5−50 μg/mL, 1−10 μg/cm2), or CuCl2 (63−630 μM, corresponding to a copper content of 5−50 μg/mL CuO in the case of complete dissolution) was applied to 96-well plates. After the appropriate incubation time under cell culture conditions (37 °C and 5% CO2), 10 μL of H2O was added to each well, and the plates © 2013 American Chemical Society
Figure 1. Absorbance of CuO NP in DMEM/FCS.
Next, an inherent reducing capacity of DMEM/FCS as well as CuO NP, CuO MP, or CuCl2 suspensions toward WST-8 was determined. Thus, DMEM/FCS, CuO MP, CuO NP (5− 50 μg/mL, 1−10 μg/cm 2 ), or CuCl 2 (63−630 μM, corresponding to a copper content of 5−50 μg/mL CuO in the case of complete dissolution) suspensions were incubated for 4 h under cell culture conditions before the addition of 10 μL of CCK-8 to each well. After 1 h, the absorbance was Received: November 7, 2013 Published: December 31, 2013 169
dx.doi.org/10.1021/tx400414c | Chem. Res. Toxicol. 2014, 27, 169−171
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manuscript submitted for publication). Thus, interference of redox-active copper ions with the reduction of WST-8 was considered, and further experiments were conducted with CuCl2. To elucidate a potential reduction-inhibiting effect of copper ions, A549 cells were treated with CuCl2 (1−250 μM) and simultaneously with CCK-8 reagent. After incubation for 1 h, the absorbance was measured at 450 nm (for the detailed procedure, see the Supporting Information). As shown in Figure 3, a strong apparent loss of the metabolic activity by
measured at 450 nm (see the detailed procedure in the Supporting Information). No increase in absorbance and thus no WST-8 reduction were detected (data not shown). Consequently, the method was considered suitable when correcting for the intrinsic absorbance of the CuO NP incubation suspension. Thus, for the calculation of the final absorption, mean values of the intrinsic absorption controls of every incubation suspension were subtracted. Subsequently, the impacts on the metabolic activity and cytotoxicity of CuO NP, CuO MP, and CuCl2 were determined, comparing the CCK-8 assay, CN, and CFA after treatment of A549 cells with the respective copper compound for 4 h. For the assessment of WST-8 reduction, 10 μL of CCK-8 reagent was added to each well of the 96-well plate; wells for the intrinsic absorbance controls contained cell-free particle suspensions and were supplied with 10 μL of H2O instead of CCK-8. For CN and CFA assessment, cells were electronically counted (CN) after being incubated, and 300 cells were reseeded in fresh DMEM/FCS for CFA; after postincubation for 7−10 days, colonies were fixed, stained, and counted (for details, see the Supporting Information). CuO MP did not show any alteration of the metabolic activity or cytotoxicity in the test systems applied (data not shown). Also, none of the substances decreased CN (Figure 2A,B). With
Figure 3. Impact of different incubation protocols on the apparent decrease in the metabolic activity by CuCl2 applying the CCK-8 reagent.
CuCl2 was detected starting at 10 μM CuCl2 (68% of the control). However, results were different when a modified procedure was applied, i.e., the CuCl2 suspensions were added to A549 cells for 1 h, and they were removed from the monolayer before the addition of CCK-8 reagent for an additional 1 h. Here, no decrease in absorbance was detected up to 250 μM CuCl2, indicating that the apparent decrease in metabolic activity provoked by CuCl2 in the CCK-8 assay was a false positive result. Assuming that the proposed mechanism of extracellular WST-1 reduction can be applied to WST-8, reducing equivalents from the mitochondrial electron transport chain are transferred through the cytosol to the cellular membrane in the form of NADH.2 The electrons cross the membrane by trans-plasma membrane electron transport (tPMET) before they are transferred to mPMS. The radical mPMS• then participates in WST-8 reduction, resulting in the formation of the respective formazan. Copper ions might interfere at several points of the reaction chain starting at the cell membrane. Thus, tPMET is believed to involve the participation of a trans-plasma membrane NADH oxidase.10 Because copper ions have a high affinity for redoxsensitive structures like sulfhydryl groups in proteins, the NADH oxidase activity and therefore tPMET might be altered or inhibited, leading to a potentially diminished level of WST-8 reduction. If the NADH oxidase activity remains unchanged, electrons might be captured before they reach mPMS because of the higher redox potential of the copper redox couple (0.15 V) compared to that of the mPMS redox couple (0.07 V). Finally, if the radical forms properly, then copper ions might oxidize mPMS• before the radical reaches the WST-8 molecule. In summary, the data presented in this study demonstrate for the first time that copper ions interfere with the reduction of WST-8 and generate a false positive result by overestimating the reduction of metabolic activity. As a consequence, the original application procedure given by the manufacturer was improved by separating the copper incubation from the CCK-8 reagent by an intermediate washing step. Thus, the false positive result was eliminated. However, while this modified
Figure 2. Impact of (A) CuO NP and (B) CuCl2 on metabolic activity (WST-8 reduction), cell number (CN), and colony forming ability (CFA) after incubation for 4 h in A549 cells.
regard to CFA, only CuO NP caused a concentrationdependent decrease starting from 5 μg/mL (Figure 2A). However, treatments with CuO NP or CuCl2 resulted in distinct decreases in the level of WST-8 reduction (Figure 2A,B). The effect was most pronounced after treatment with CuCl2 starting at the lowest concentration of 63 μM (53% of the control) and reaching 3% of the control at 252 μM (Figure 2B). Altogether, the distinct apparent loss of metabolic activity especially provoked by CuCl2 and to a lesser extent by CuO NP, as determined by the CCK-8 assay, was not reflected by a decrease in CN or CFA. Interestingly, the extent of the overestimated decrease in metabolic activity resembled the order of solubility of the copper compounds in DMEM/FCS (A. Semisch et al., 170
dx.doi.org/10.1021/tx400414c | Chem. Res. Toxicol. 2014, 27, 169−171
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interference with assay processes and components of classic in vitro tests. Nanotoxicology, DOI: 10.3109/17435390.2013. (9) Herzog, E., Casey, A., Lyng, F. M., Chambers, G., Byrne, H. J., and Davoren, M. (2007) A new approach to the toxicity testing of carbon-based nanomaterials: The clonogenic assay. Toxicol. Lett. 174, 49−60. (10) Löw, H., Crane, F. L., and Morré, D. J. (2012) Putting together a plasma membrane NADH oxidase: A tale of three laboratories. Int. J. Biochem. Cell Biol. 44, 1834−1838.
approach may be useful for investigating water-soluble metal compounds, it will not exclude artifacts in the case of only partly water-soluble nano- or microparticulate metal compounds because these can hardly be removed completely from cells. Therefore, the general application of the colorimetric CCK-8 assay appears to be questionable, and it is of utmost importance either to carefully check for interference or to rely on less error-prone methods like CN or CFA.
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ASSOCIATED CONTENT
S Supporting Information *
Particle characteristics and experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Funding
This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Exzellenzinitative KIT. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS CCK-8, Cell Counting Kit-8; CFA, colony forming ability; CuCl2, copper chloride; CN, cell number; CuO MP, copper oxide microparticles; CuO NP, copper oxide nanoparticles; DMEM/FCS, Dulbecco’s modified Eagle’s medium supplemented with 10% FCS; mPMS, 1-methoxy-5-methylphenazinium methyl sulfate; tPMET, trans-plasma membrane electron transport; WST-8, water-soluble tetrazolium-8-[2-(2-methoxy4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] monosodium salt
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REFERENCES
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