DETERMINANTS OF MTT REDUCTION IN RAT HEPATOCYTES

The determinants of reduction of the dye MTT [4,5-dimethylthiazol-2-yl ]-2,5-diphenyltetrazolium bromide) in rat hepatocytes have been invest igated. NADH, NADPH, and succinate were substrates for MTT reduction i n rat liver homogenate, activity being greatest with NADH and least wi th succinate. Similar results were obtained with submitochondrial part icles isolated from rat liver, NAD(P)H-dependent reduction of MTT was also detected in rat liver microsomes and cytosol. Rotenone, at a conc entration that inhibited NAD(P)H-dependent MTT reduction in sub-mitoch ondrial particles, did not inhibit MTT reduction in rat hepatocytes. M alonate, at a concentration that inhibited succinate-dependent MTT red uction in liver homogenate, did not inhibit MTT reduction in rat hepat ocytes. Incubation of rat hepatocytes with ethanol or lactate (increas e NADH levels), dicoumarol (inhibitor of DT-diaphorase), aminopyrine o r hexobarbitone (substrates for the NADPH-requiring cytochrome P450-de pendent microsomal monooxygenase) led to significant increases in the level of cellular MTT reduction. From these data, it is concluded that extra-mitochondrial NAD(P)H is the principal reductant for MTT reduct ion in rat hepatocytes, with mitochondrial dehydrogenase activity bein g only a minor contributor. It is also possible that cellular generati on of superoxide (as might be expected on redox cycling of endogenous quinones following inhibition of DT diaphorase by dicoumarol) may be a nother source of MTT reduction. Caution should be exercised in ascribi ng an alteration in the level of cellular MTT reduction to a change in mitochondrial performance in the absence of corroborating evidence.