Rees R S, Kingman G J, Cashmer B, Gilmont R R, Reeves C, Welsh M J, Smith D J
Department of Surgery, University of Michigan, Taubman Center, Ann Arbor 48109-0340.
J Surg Res. 1994 Apr;56(4):326-30. doi: 10.1006/jsre.1994.1050.
DT diaphorase is a flavoprotein that enzymatically transfers two electrons from quinones as intermediate substrates and has been reported to increase its activity in the liver after exposure to toxicants. In this series of experiments, we tested the hypothesis that DT diaphorase also increases its activity after exposure to oxidants following gradient ischemia in skin. Using dorsal rat flaps, oxidant stress was induced immediately or during a 7-day period of preconditioning as a bipedicle flap before the distal attachment was divided. DT diaphorase activity (delta Abs/min/100 g) or expression of message was measured during the period of preconditioning to determine the relationship between skin 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 but there was as 50-fold increase in DT diaphorase activity at the distal end 24 hr after they were divided (P < 0.05). Maximal enzyme induction of DT diaphorase activity occurred after 4 days of preconditioning and correlated with the maximal expression of mRNA. These studies provide 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.
DT 黄递酶是一种黄素蛋白，它能作为中间底物从醌类物质中酶促转移两个电子，据报道，接触毒物后其在肝脏中的活性会增加。在这一系列实验中，我们检验了这样一个假设：在皮肤梯度缺血后接触氧化剂，DT 黄递酶的活性也会增加。使用大鼠背部皮瓣，在远端附着点切断前，立即或在作为双蒂皮瓣的 7 天预处理期内诱导氧化应激。在预处理期间测量 DT 黄递酶活性（吸光度变化/分钟/100 克）或信使表达，以确定皮肤存活、酶活性和信使表达之间的关系。急性皮瓣远端有 4.7±0.8 厘米的皮肤坏死，而预处理皮瓣在远端附着点切断后没有皮肤坏死。在急性皮瓣中，最初 6 小时内整个皮瓣的 DT 黄递酶活性相等。缺血 24 小时后，皮瓣近端的 DT 黄递酶活性（1.83±0.21 吸光度变化/分钟/100 克）显著高于远端（0.005±0.01 吸光度变化/分钟/100 克），差异有统计学意义（P<0.05）。在预处理皮瓣中，酶活性没有增加，但在切断后 24 小时，远端的 DT 黄递酶活性增加了 50 倍（P<0.05）。DT 黄递酶活性的最大酶诱导发生在预处理 4 天后，且与 mRNA 的最大表达相关。这些研究提供了首个证据，表明氧化应激后 DT 黄递酶活性是可诱导的。数据还表明，DT 活性在缺血至少 6 小时内仍保持升高，可能是缺血皮肤中抗氧化活性的潜在来源。
