Comparison of alamar blue and MTT assays for high through-put screening

Abstract

The performance of alamar blue and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cell viability assays in a high through-put format were compared. A total of 117 drugs chosen for their wide range of therapeutic areas were screened at 10 μM using both assays in human hepatoma cell line HepG2. Except for terfenadine and astemizole, which performed consistently in both assays, the alamar blue assay was slightly more sensitive than the MTT assay for most compounds. The MTT assay was less sensitive detecting an effect for daunorubicin and trifluoperazine. Seven drugs, astemizole, daunorubicin, ellipticine, fluphenazine, terfenadine, thioridazine and trifluoperazine, had percent viability results of 55% or less in the alamar blue assay at the single point screen. These were re-tested in both assays for reconfirmation of cytotoxicity and determination of the EC₅₀ values. Except for daunorubicin, the EC₅₀ values were comparable in both assays. Based on these results and the Z^′-factor assessment of assay quality, both assays provided useful information to identify in vitro cytotoxic drugs at early stages of drug candidate selection. However, careful interpretation of data is warranted due to the possibility of false positive or negative results caused by inducers and/or inhibitors of metabolic enzymes that are responsible for transformation of cell toxicity end points, as we demonstrated using dicumarol.

Introduction

The study of drug-like properties of new chemical entities (NCEs) or lead candidates during the discovery process has gained substantial momentum in the past few years due to a high rate of drug candidate failures during clinical trials. Some of those failures are due to non-clinical toxicity and adverse side effects. The inclusion of in vitro absorption, distribution, metabolism, elimination and toxicity (ADME/Tox) studies in the discovery process provides a promising strategy to decrease the number of late drug candidate failures. The toxicity of a drug is a critical property and is the most unpredictable characteristic of a drug because it can be species and organ-specific (Ponsoda et al., 1995). Assessment of a compound's toxicity to various cell types can be made using in vitro cytotoxicity tests, which are available and widely used. These assays have a benefit of making the process of screening large chemical libraries easier.

This paper compares the performance of alamar blue and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assays as an index of intrinsic cytotoxicity. A total of 117 known pharmaceutical compounds were screened in both assays in a 96-well plate high through-put screening (HTS) format utilizing HepG2 cells, a human hepatoma cell line. The MTT and alamar blue assays test for intrinsic cytotoxicity using different mechanisms. The MTT assay is a well-documented cell viability assay and has been modified by several investigators since it was first developed by Mosmann (1983). It is believed to be based on the transformation of tetrazolium salt by mitochondrial succinic dehydrogenases in viable cells yielding purple formazan crystals that are not soluble in aqueous solution. Alamar blue is a sensitive oxidation–reduction indicator that fluoresces and changes color upon reduction by living cells. The reduction of alamar blue is believed to be mediated by mitochondrial enzymes (O'Brien et al., 2000). However, recent data suggest that cytosolic and microsomal enzymes also contribute to the reduction of alamar blue and MTT (Gonzalez and Tarloff, 2001). Also it has been shown that only 25–40% of MTT-formazan deposits were associated with mitochondria in HepG2 cells (Bernas and Dobrucki, 2002).

The quality of both assays was assessed using the Z^′-factor, a parameter for the quality of an assay in the absence of test compounds. An assay associated with a Z^′-factor approaching 1 is considered reproducible (Zhang et al., 2002), while an assay with a Z^′-factor of a negative value or a value near zero is not suitable for HTS and indicates that the assay requires further optimization.