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

Alamar blue and MTT are indicators used to measure cytotoxicity of various chemicals in cultured cells. Both Alamar blue and MTT are reduced by mitoch ondrial enzymes. We observed enhanced fluorescence of Alamar blue when kidn ey epithelial cells were co-incubated with hepatic post-mitochondrial super natant (S9) fractions as compared with cells incubated in the absence of S9 fractions. The present studies were carried out to determine whether hepat ic cytosolic and/or microsomal enzymes were capable of metabolizing Alamar blue and/or MTT to their reduced products. Livers from female Sprague-Dawle y rats were used to prepare S9 fraction, and mitochondrial, microsomal and cytosolic fractions. Fractions containing 1 or 5 mg protein/ml were incubat ed with Alamar blue or MTT for up to 4 h. Fluorescence (Alamar blue) or abs orbance (MTT) were determined and expressed as differences between treated wells and controls. Hepatic fractions (S9, mitochondria, microsomes and cyt osol) caused concentration- and time-dependent increases in Alamar blue flu orescence and MTT absorbance. Reduction of Alamar blue and MTT by hepatic S 9 fraction was abolished by heating. Reduction of Alamar blue by hepatic mi tochondria was approximately equivalent to that catalyzed by hepatic S9 fra ction or cytosol. Reduction of MTT by hepatic mitochondria was approximatel y equivalent to that catalyzed by hepatic S9 fraction or microsomes. These data indicate that mitochondrial, cytosolic and microsomal enzymes reduce A lamar blue and MTT. Therefore, caution should be exercised in ascribing dec reases in viability as due solely to mitochondrial damage when using either of these dyes. (C) 2001 Elsevier Science Ltd. All rights reserved.