Evaluation of hepatic subcellular fractions for Alamar blue and MTT reductase activity

Abstract

Alamar blue and MTT are indicators used to measure cytotoxicity of various chemicals in cultured cells. Both Alamar blue and MTT are reduced by mitochondrial enzymes. We observed enhanced fluorescence of Alamar blue when kidney epithelial cells were co-incubated with hepatic post-mitochondrial supernatant (S9) fractions as compared with cells incubated in the absence of S9 fractions. The present studies were carried out to determine whether hepatic cytosolic and/or microsomal enzymes were capable of metabolizing Alamar blue and/or MTT to their reduced products. Livers from female Sprague–Dawley rats were used to prepare S9 fraction, and mitochondrial, microsomal and cytosolic fractions. Fractions containing 1 or 5 mg protein/ml were incubated with Alamar blue or MTT for up to 4 h. Fluorescence (Alamar blue) or absorbance (MTT) were determined and expressed as differences between treated wells and controls. Hepatic fractions (S9, mitochondria, microsomes and cytosol) caused concentration- and time-dependent increases in Alamar blue fluorescence and MTT absorbance. Reduction of Alamar blue and MTT by hepatic S9 fraction was abolished by heating. Reduction of Alamar blue by hepatic mitochondria was approximately equivalent to that catalyzed by hepatic S9 fraction or cytosol. Reduction of MTT by hepatic mitochondria was approximately equivalent to that catalyzed by hepatic S9 fraction or microsomes. These data indicate that mitochondrial, cytosolic and microsomal enzymes reduce Alamar blue and MTT. Therefore, caution should be exercised in ascribing decreases in viability as due solely to mitochondrial damage when using either of these dyes.

Introduction

Alamar blue and MTT are indicators used to measure cytotoxicity of various chemicals in cultured cells (Takeuchi, Bada and Shigeta, 1991, Sun, Wu and Last, 1995, Schirmer et al., 1998, Slaughter et al., 1999, Born, Hu and Lehman-McKeeman, 2000). The use of Alamar blue is based on the ability of cellular enzymes to reduce the non-fluorescent blue dye to a pink fluorescent compound, allowing detection of cell growth by spectrofluorometry and spectrophotometry (de Fries et al., 1995). Enzymes that may be involved in Alamar blue reduction include mitochondrial enzymes such as flavin mononucleotide dehydrogenase, flavin adenine dinucleotide dehydrogenase, nicotinamide adenine dinucleotide dehydrogenase, nicotinamide adenine phosphate dehydrogenase and cytochromes (O'Brien et al., 2000). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) is also used to measure viability in cultured cells. MTT is reduced by mitochondrial dehydrogenases, which cleave the yellow tetrazolium salt MTT to purple formazan crystals (Mosmann, 1983). Chemically-induced decreases in Alamar blue fluorescence and MTT absorbance are usually interpreted as indicative of mitochondrial damage. However, cells contain numerous enzymes capable of chemical reduction reactions, including cytosolic and microsomal enzymes.

We observed enhanced fluorescence of Alamar blue when kidney epithelial cells were co-incubated with hepatic post-mitochondrial supernatant (S9) fractions as compared with cells incubated in the absence of S9 fractions. The present studies were carried out to determine whether hepatic cytosolic and/or microsomal enzymes were capable of metabolizing Alamar blue and/or MTT to their reduced products.