DT DIAPHORASE - INCREASED ENZYME-ACTIVITY AND MESSENGER-RNA EXPRESSION IN OXIDANT STRESS OF SKIN

DT diaphorase is a flavoprotein that enzymaticly transfers two electro ns from quinones as intermediate substrates and has been reported to i ncrease its activity in the liver after exposure to toxicants. In this series of experiments, we tested the hypothesis that DT diaphorase al so increases its activity after exposure to oxidants following gradien t ischemia in skin. Using dorsal rat flaps, oxidant stress was induced immediately or during a 7-day period of preconditioning as a bipedicl e flap before the distal attachment was divided. DT diaphorase activit y (Delta Abs/min/100 g) or expression of message was measured during t he period of preconditioning to determine the relationship between ski n survival, enzyme activity, and expression of message. There was 4.7 +/- 0.8 cm of skin necrosis in the distal end of acute flaps while the preconditioned flaps had no skin necrosis after the distal attachment was divided. In the acute flaps, the DT diaphorase activity was equal throughout the flap for the first 6 hr. After 24 hr of ischemia, the DT diaphorase activity was significantly higher in the proximal end of the flap (1.83 +/- 0.21 Delta Abs/min/100 g) than that in the distal end (0.005 +/- 0.01 Delta Abs/min/100 g), which was significant (P < 0 .05). In the preconditioned flaps, enzyme activity did not increase bu t there was as 50-fold increase in DT diaphorase activity at the dista l end 24 hr after they were divided (P < 0.05). Maximal enzyme inducti on of DT diaphorase activity occurred after 4 days of preconditioning and correlated with the maximal expression of mRNA. These studies prov ide the first evidence that DT diaphorase enzyme activity is inducible after oxidant stress. The data also suggests that DT activity remains elevated for at least 6 hr of ischemia and may be a potential source of anti-oxidant activity in ischemic skin. (C) 1994 Academic Press, In c.