Kerver E D, Vogels I M, Bosch K S, Vreeling-Sindelárová H, Van den Munckhof R J, Frederiks W M
Academic Medical Centre, University of Amsterdam, The Netherlands.
Histochem J. 1997 Mar;29(3):229-37. doi: 10.1023/a:1026453926517.
In the present study, the endogenous formation of reactive oxygen species was localized in rat liver and small intestine. The 3,3'-diaminobenzidine (DAB)-Mn2+ technique in which cobalt ions were included in the incubation medium was applied to unfixed cryostat sections of intact tissues. Addition of manganese ions to the DAB-Co(2+)-containing medium greatly increased the amounts of final reaction product formed compared with incubations with only DAB and cobalt ions. In liver, a blue final reaction product was deposited, particularly in hepatocytes surrounding portal tracts. In the small intestine, the DAB-cobalt complex was mainly found at the basal side of enterocytes. Goblet cells remained unstained. Electron microscopical images revealed that an electron-dense reaction product was exclusively present at both inner and outer membranes and at the intermembrane space in mitochondria of liver parenchymal cells and duodenal enterocytes. It was shown that the spontaneous formation of final reaction product was enzymatic and dependent on the presence of oxygen in the medium. Sulphide decreased the reaction, which may indicate that cytochrome c oxidase was partially involved. Benzoquinone and histidine, which are scavengers of superoxide anions and singlet oxygen respectively, reduced the amount of final reaction product considerably. Furthermore, the formation of final reaction product was sensitive to specific inhibitors of NADH:coenzyme Q reductase and aldehyde oxidase, indicating that these enzymes were at least partly responsible for the generation of superoxide anions and singlet oxygen and for the formation of the DAB-cobalt complex.
在本研究中，活性氧的内源性形成定位于大鼠肝脏和小肠。将含有钴离子的3,3'-二氨基联苯胺（DAB）-Mn2+技术应用于完整组织的未固定低温恒温器切片。与仅用DAB和钴离子孵育相比，向含DAB-Co(2+)的培养基中添加锰离子大大增加了最终反应产物的生成量。在肝脏中，蓝色的最终反应产物沉积，特别是在门静脉周围的肝细胞中。在小肠中，DAB-钴复合物主要存在于肠上皮细胞的基底侧。杯状细胞未被染色。电子显微镜图像显示，电子致密反应产物仅存在于肝实质细胞和十二指肠肠上皮细胞线粒体的内膜和外膜以及膜间隙。结果表明，最终反应产物的自发形成是酶促反应，并且依赖于培养基中氧气的存在。硫化物降低了反应，这可能表明细胞色素c氧化酶部分参与其中。分别作为超氧阴离子和单线态氧清除剂的苯醌和组氨酸，显著减少了最终反应产物的量。此外，最终反应产物的形成对NADH：辅酶Q还原酶和醛氧化酶的特异性抑制剂敏感，表明这些酶至少部分负责超氧阴离子和单线态氧的产生以及DAB-钴复合物的形成。
