REDUCTION OF THE NEW TETRAZOLIUM DYE, ALAMAR-BLUE(TM), IN CULTURED RAT HEPATOCYTES AND LIVER FRACTIONS

Alamar Blue(TM) (AB) is a new tetrazolium dye substrate that has been introduced as an alternative cell viability indicator. AB is reduced b y intracellular reductases to a product which is exported from cells a nd can be quantified by fluorescent or spectrophotometric methods. We investigated the processes by which AB was reduced in liver cytosolic, microsomal or mitochondrial fractions and in cultured rat hepatocytes . AB reduction was catalysed by all liver fractions in an NADPH-depend ent and NADH-dependent manner; the cytosolic fraction catalysed the hi ghest rate of AB reduction. All of these activities were inhibited by dicoumarol (10 mu M), except AB reduction catalysed by NADH in mitocho ndrial fractions, which was resistant to the effects of dicoumarol and the metabolic inhibitors, but sensitive to inhibition by mercury (II) chloride. In hepatocyte cultures, AB reduction was stimulated by dico umarol (10 mu M), menadione (10 mu M), rotenone (10 mu M), lactate (1- 10mM) and fluoride (3-10mM). Potassium cyanide, ethanol and malonate h ad little effect. The results from this study suggest that AB is reduc ed in an NADPH-quinone oxidoreductase-dependent fashion, but that supe roxide may also be involved in the reduction of AB. The modulation of AB reduction by lactate means that AB reduction may be modified by alt erations in intermediary metabolism which are not a reflection of cell lethality. Therefore, great care should he exercised when using AB re duction as a viability indicator.