ASCORBATE IS THE PRIMARY REDUCTANT OF THE PHENOXYL RADICAL OF ETOPOSIDE IN THE PRESENCE OF THIOLS BOTH IN CELL HOMOGENATES AND IN MODEL SYSTEMS
ASCORBATE IS THE PRIMARY REDUCTANT OF THE PHENOXYL RADICAL OF ETOPOSIDE IN THE PRESENCE OF THIOLS BOTH IN CELL HOMOGENATES AND IN MODEL SYSTEMS
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DOI：
10.1021/bi00198a034
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发表时间：
1994-08-16
期刊：
影响因子：
2.9
通讯作者：
STOYANOVSKY, DA
中科院分区：
文献类型：
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作者：
KAGAN, VE;YALOWICH, JC;STOYANOVSKY, DA
苯氧基自由基是酚类化合物氧化成醌式衍生物（醌、醌甲基化物）的中间体，已知其通过直接与大分子靶点相互作用或通过产生毒性活性氧物质而充当最终致突变剂、致癌剂和细胞毒性剂。苯氧基自由基的单电子还原可以逆转酚类化合物氧化活化为醌类化合物，从而防止其细胞毒性作用。在目前的工作中，我们研究了抗坏血酸，硫醇（谷胱甘肽，二氢硫辛酸和金属硫蛋白），及其组合与VP-16 [依托泊苷，4 '-去甲基表鬼臼毒素-9-（4，6-O-亚乙基-β-D-吡喃葡萄糖苷）]，一种受阻酚广泛用作抗肿瘤药物的酪氨酸酶催化氧化产生的苯氧基自由基的相互作用。我们发现，通过液相色谱-离子喷雾质谱和电子自旋共振（ESR），酪氨酸酶引起氧化VP-16其邻醌和芳构化衍生物通过中间体形成的苯氧基自由基。抗坏血酸和硫醇（GSH，二氢硫辛酸和金属硫蛋白）都能够直接减少VP-16苯氧基自由基，并防止其氧化。还原剂可猝灭VP-16苯氧自由基的特征ESR信号。在存在抗坏血酸的情况下，检测到semidehydroascorbic自由基ESR信号;硫醇在ESR谱中不产生信号。在组合中，抗坏血酸加GSH和抗坏血酸加金属硫蛋白在减少VP-16苯氧基自由基中独立和相加地起作用。抗坏血酸是更具反应性的：VP-16依赖的氧化GSH或金属硫蛋白开始后，抗坏血酸完全氧化。半氢抗坏血酸自由基的ESR信号先于GSH和金属硫蛋白对VP-16苯氧基自由基的猝灭。在抗坏血酸加二氢硫辛酸的存在下，抗坏血酸也是对VP-16苯氧基自由基比二氢硫辛酸更反应性，但抗坏血酸浓度保持在其再生从脱氢抗坏血酸二氢硫辛酸的费用。在ESR谱中，半氢抗坏血酸自由基的ESR信号连续检测，然后突然被VP-16苯氧基自由基信号取代。当VP-16和酪氨酸酶在视网膜或肝细胞匀浆的存在下孵育时，通过ESR观察到VP-16自由基信号出现的两相滞后期：抗坏血酸依赖性部分（可观察到的半氢抗坏血酸自由基，对抗坏血酸氧化酶敏感）和巯基依赖性部分（光谱中无自由基信号，对汞水杨酸敏感）。约50%的硫醇依赖的滞后期的部分可以占内源性GSH（如所揭示的治疗与GSH过氧化物酶+枯烯氢过氧化物）。匀浆防止VP-16被酪氨酸酶氧化。抗坏血酸和硫醇，两个主要的水溶性细胞内抗氧化剂，直接减少苯氧基自由基的能力可能是一个重要的机制，其保护功能对酚类/醌氧化还原对细胞毒性。
Phenoxyl radicals are intermediates in the oxidation of phenolic compounds to quinoid derivatives (quinones, quinone methides), which are known to act as ultimate mutagenic, carcinogenic, and cytotoxic agents by directly interacting with macromolecular targets or by generating toxic reactive oxygen species. One-electron reduction of phenoxyl radicals may reverse oxidative activation of phenolic compounds to quinoids, thus preventing their cytotoxic effects. In the present work, we studied interactions of ascorbate, thiols (glutathione, dihydrolipoic acid, and metallothioneins), and combinations thereof with the phenoxyl radical generated by tyrosinase-catalyzed oxidation of VP-16 [etoposide, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyranoside) ], a hindered phenol widely used as an antitumor drug. We found by liquid chromatography-ionspray mass spectrometry and electron spin resonance (ESR) that tyrosinase caused oxidation of VP-16 to its o-quinone and aromatized derivative via intermediate formation of the phenoxyl radical. Both ascorbate and thiols (GSH, dihydrolipoic acid, and metallothioneins) were able to directly reduce the VP-16 phenoxyl radical and prevent its oxidation. The characteristic ESR signal of the VP-16 phenoxyl radical was quenched by the reductants. The semidehydroascorbyl radical ESR signal was detected in the presence of ascorbate; thiols did not produce signals in the ESR spectra. In combinations, ascorbate plus GSH and ascorbate plus metallothionein acted independently and additively in reducing the VP-16 phenoxyl radical. Ascorbate was more reactive: the VP-16-dependent oxidation of GSH or metallothionein commenced only after complete oxidation of ascorbate. The semidehydroascorbyl radical ESR signal preceded the quenching of the VP-16 phenoxyl radical by GSH and metallothionein. In the presence of ascorbate plus dihydrolipoic acid, ascorbate was also more reactive toward the VP-16 phenoxyl radical than dihydrolipoic acid, but the ascorbate concentration was maintained at the expense of its regeneration from dehydroascorbate by dihydrolipoic acid. In ESR spectra, the semidehydroascorbyl radical ESR signal was continuously detected and then was abruptly substituted by the VP-16 phenoxyl radical signal. When VP-16 and tyrosinase were incubated in the presence of retina or hepatocyte homogenates, a two-phase lag period was observed by ESR for the appearance of the VP-16 radical signal: an ascorbate-dependent part (semidehydroascorbyl radical observable, sensitive to ascorbate oxidase) and thiol-dependent part (no radical signals in the spectra, sensitive to mersalyl acid). About 50% of the thiol-dependent part of the lag period could be accounted for by endogenous GSH (as revealed by treatment with GSH peroxidase + cumene hydroperoxide). Homogenates prevented VP-16 oxidation by tyrosinase. The ability of ascorbate and thiols, the two major water-soluble intracellular antioxidants, to directly reduce phenoxyl radicals may be an important mechanism of their protective function against cytotoxicity of phenolic/quinoid redox couples.