CriticalAppraisaloftheMTTAssayinthePresenceofRottlerin6

Our experience indicates that it may not be sufficient to change the medium containing Rottlerin and to wash the cells before adding MTT to avoid a potential bias in concluding results. In fact, the Rottlerin lipophylic nature allows the molecule to freely cross the plasma membrane, to accumulate inside the cell, to target mitochondria, and to exert the well documented uncoupling effects (4). The Rottlerin intracellular accumulation can be easily verified by a simple observation: The cells remain yellow-colored even after washing.

The results presented in the current paper also indicate that it may not be sufficient to include a control without cells in the MTT assay, a stratagem that has been recommended to avoid false-positive results in the presence of molecules with reducing properties, such as flavonoids (18). Rottlerin, indeed, does not react with MTT in vitro, but causes enhanced MTT reduction in cultured cells.

Similar to the MTT, the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) measures cell viability based on the activity of mitochondria enzymes in live cells. Unlike the water-insoluble formazan produced from MTT, XTT is readily reduced to a highly water-soluble orange colored product, thus eliminating the need for the solubilization step required for the MTT assay.

Similar to the XTT, the Dojindo''s tetrazolium salt, the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2H tetrazolium, monosodium salt (WST-8), marketed as Cell Counting kit-8 (CCK-8), is reduced by cellular dehydrogenases, in the presence of an electron carrier, to an orange formazan product that is water-soluble.

Despite the technical differences and the detection sensitivity, the MTT, XTT, and WST-8 work on exactly the same principle and measure the same parameter. Therefore, it can be anticipated that the uncoupling properties of Rottlerin also could have a detrimental effect on the results from both XTT and WST-8 cell proliferation assays.

On the basis of our findings, it can be suggested that a safety alternative test in toxicological studies could be the detection of LDH released in the medium by cultured cells, since no interference was observed in our LDH assay in the presence of Rottlerin in the most widely used concentration range (5–20 μM). Some other LDH commercial kits are based on a two steps reaction: (1) the oxidation of lactate to pyruvate and (2) reduction of MTT to formazan by pyruvate. Also these LDH assays can be recommended in the presence of Rottlerin, the drug did not exhibit any direct reactivity toward MTT salts in vitro; thus no interference should be expected.

Moreover, we also discourage the use of the MTT assay in the presence of mitochondrial uncoupling agents; a number of lipophylic molecules, both natural and synthetic, such as polyphenols and steroid hormones, have uncoupling properties. In these cases, the direct cell counting or the direct measurement of cell proliferation (tritiated thymidine or others) seems to be more appropriate than the MTT assay.

Another widely used method to assess proliferation, cytotoxicity, or viability of cultured mammalian cells and to monitor the effects of a wide range of drugs and biological compounds is the measurement of ATP content in lysed cells by colorimetric, fluorometric, and radioisotopic assays. ATP is a good marker for cell viability because it is present in all metabolically active cells, and the concentration declines very rapidly when the cells undergo necrosis or apoptosis. However, although we have not verified a possible interference by Rottlerin and other uncouplers in ATP detection by the methods above, it is possible, or better obvious, that the decrease in ATP caused by mitochondrial uncoupling could be erroneously interpreted as a decrease in cell viability (false or enhanced negative result). We have no suggestions on how to circumvent the interference in these cases and can only discourage the use of ATP measurement in the presence of known or putative uncoupling molecules.

In closing, we have demonstrated in this report that, although the MTT method is the most frequently used in the assessment of the viable cell number, its inadequacy is evident in presence of drugs/compound that cause alterations in mitochondria activity. We observed a significant pitfall in the presence of the chemicals tested, resulting in inaccurate cell number estimation and misinterpretation of results. Therefore, we suggest caution in the use of the MTT assay as a proliferation/viability/toxicity test in the presence of Rottlerin and uncoupling agents in general, especially if not corroborated using alternative/complementary assays.

Acknowledgment  This study was supported by research projects grants from “Fondazione Monte dei Paschi di Siena” (2008).

By: Emanuela Maioli1 , Claudia Torricelli1, Vittoria Fortino1, Filippo Carlucci2, Valentina Tommassini2, Adriana Pacini1

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