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.