Assessment of antileishmanial and cytotoxic activities of some phenolic acids using the MTT assay – a critical evaluation

The MTT assay, based on the reduction of a tetrazolium salt into a blue formazan product by dehydrogenase activity in viable cells, is widely used for measuring cell viability. Problems with MTT in the presence of plant phenolic acids and related reports [1] prompted the critical re-evaluation of a method that we routinely use for screening antileishmanial compounds and evaluating their potential toxicity for host cells. For this, we used caffeic, benzoic, p-hydroxybenzoic, and gallic acid, and the methyl and ethyl esters of gallic acid.

We show that gallic acid and its tested esters could reduce MTT in the absence of living cells. In a cell viability assay, this apparent metabolic effect would lead to false positive results. Also, serum proteins interfered with the samples in a time-dependent manner. For example, prolonged pre-incubation of gallic acid in medium reduced its cytotoxicity (RAW 264.7 cells; IC₅₀ of 230µM at 30min → 380µM at 7h). Further, when a standardized cell suspension was added to the sample, the IC₅₀ values of some phenolics were conspicuously smaller compared to when the samples were given to an existing cell monolayer. Parallel investigations by FACS analysis confirmed these findings. For testing antileishmanial activity with the MTT assay, a starting concentration of 2×10⁵ parasites/ well was found useful. No significant differences in sensitivity were seen with L. donovani and L. major.

We conclude that cell-free controls are essential when cell viability is to be tested. On the other hand, in our assay for antileishmanial activity against intracellular amastigotes [2], samples are fully removed before host cell lysis and addition of MTT, thus rendering this assay much less prone to irregular results.

References: 1. Rollino, C. et al. (1995), J. Immunol. Methods 185: 141–143. 2. Kiderlen, A.F., Kaye, P.M. (1990), J. Immunol Methods 127: 11–18.