1,3-Dinitrobenzene metabolism and protein binding.
1,3-Dinitrobenzene metabolism and protein binding.
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DOI：
10.1021/tx015554
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发表时间：
2002-03
影响因子：
4.1
通讯作者：
Ian T Reeve;Marion G. Miller
中科院分区：
文献类型：
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作者：
Ian T Reeve;Marion G. Miller
1，3-二硝基苯是一种睾丸毒物，会在大鼠的生精小管上产生损伤。在目前的研究中，我们研究了生精小管的哪些亚细胞部分能够进行1，3-二硝基苯的代谢和蛋白质加合物的形成。肝脏的亚细胞部分被用作阳性对照，以进一步研究潜在的重要结合蛋白。用200微米[(14)C]1，3-二硝基苯和2 mM NADH或NADPH孵育每个组织的微粒体、胞浆和线粒体。由于硝化还原是一种对氧敏感的代谢途径，因此在有氧和无氧的情况下进行培养。在厌氧条件下，1，3-二硝基苯被代谢为硝基苯胺和/或硝基苯羟胺。在有氧条件下，代谢物的形成受到抑制，这表明存在一个氧依赖的氧化还原循环。生精小管在有氧条件下不产生代谢产物。在没有氧气的情况下，只有线粒体产生1，3-二硝基苯的代谢物。对于肝脏，在厌氧条件下，所有三个亚细胞组分都会产生1，3-二硝基苯的代谢物，其中微生物体的活性最高。然而，在有氧条件下，只有微生物体产生代谢物。一维凝胶电泳法显示，肝脏和生精小管亚细胞组分中蛋白质加合物的形成与代谢物的形成有关。在生精小管线粒体孵育中加入GSH可减少(14)C标记蛋白的数量。此外，当生精小管线粒体与1，3-二硝基苯以更高的蛋白质浓度孵育时，54 kDa蛋白质的放射性标记变得更加突出。肝线粒体蛋白与[(14)C]1，3-二硝基苯和NADPH孵育后的双向凝胶电泳法得到三种主要的放射性标记蛋白，大小相同(54 KDa)。氨基酸序列分析表明，这些蛋白质均为大鼠线粒体乙醛脱氢酶。
1,3-Dinitrobenzene is a testicular toxicant, which produces a lesion in the seminiferous tubules of the rat. In the present study, we investigated which subcellular fractions of the seminiferous tubules are capable of 1,3-dinitrobenzene metabolism and protein adduct formation. Subcellular fractions of the liver were used as positive controls and to further investigate potentially important binding proteins. Microsomes, cytosol, and mitochondria prepared from each tissue were incubated with 200 microM [(14)C]1,3-dinitrobenzene and 2 mM NADH or NADPH. Since nitroreduction is an oxygen sensitive metabolic pathway, incubations were carried out in the presence and absence of oxygen. Under anaerobic conditions, 1,3-dinitrobenzene was metabolized to nitroaniline and/or nitrophenylhydroxylamine. Metabolite formation was inhibited under aerobic conditions, suggesting the presence of an oxygen-dependent redox-cycle. For the seminiferous tubules, no metabolites were generated under aerobic conditions. In the absence of oxygen, only the mitochondria produced 1,3-dinitrobenzene metabolites. For the liver, under anaerobic conditions, all three subcellular fractions produced 1,3-dinitrobenzene metabolites with the microsomes containing the greatest activity. However, under aerobic conditions, only the microsomes generated metabolites. One-dimensional gel electrophoresis demonstrated that protein adduct formation within the liver and seminiferous tubule subcellular fractions correlated with metabolite formation. Addition of GSH to seminiferous tubule mitochondrial incubations decreased the amount of (14)C-labeled protein. Moreover, when seminiferous tubule mitochondria were incubated with 1,3-dinitrobenzene at an increased protein concentration, radioactive labeling of a 54 kDa protein became more prominent. Two-dimensional gel electrophoresis of liver mitochondrial protein incubated with [(14)C]1,3-dinitrobenzene and NADPH yielded three predominantly radiolabeled proteins of the same approximate size (54 kDa). Amino acid sequencing identified each of these proteins as rat mitochondrial aldehyde dehydrogenase.