Mode of action of butylated hydroxyanisole (BHA) and other phenols in preventing loss of 11 beta-hydroxylase activity in cultured bovine adrenocortical cells.
Mode of action of butylated hydroxyanisole (BHA) and other phenols in preventing loss of 11 beta-hydroxylase activity in cultured bovine adrenocortical cells.
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丁基羟基苯甲醚 (BHA) 和其他酚类在防止培养的牛肾上腺皮质细胞中 11 β-羟化酶活性丧失方面的作用模式。
DOI：
10.1016/0006-2952(85)90768-3
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发表时间：
1985
影响因子：
5.8
通讯作者：
S. E. Harris
中科院分区：
文献类型：
--
作者：
P. Hornsby;K. Aldern;S. E. Harris
当培养的牛肾上腺皮质细胞与皮质醇或其他类固醇(11β-羟基酶(细胞色素P-45011β)的假底物)孵育时，酶的活性降低。在以前的实验中，有三种物质被证明可以保护11β-羟基酶在假底物存在的情况下不丧失酶活性：BHA(丁基羟基苯甲醚，2(3)-叔丁基-4-甲氧基苯酚)，二甲基亚砜和甲孕酮。本实验考察了BHA的几种酚类类似物在该体系中的保护作用，并将它们的活性与DMSO和美曲酮的活性进行了比较。在用50βM皮质醇孵育24小时的培养的肾上腺皮质细胞中，测试了不同的BHA类似物防止11μ羟基酶活性丧失的能力时，发现苯酚本身与BHA大致相同。在苯酚中添加甲基、甲氧基和苄基并不会降低化合物的保护活性，但添加一个叔丁基尤其是两个叔丁基会大大降低化合物的保护活性。因此，与BHA相反，BHT(2，6-二叔丁基-4-甲基苯酚)是无效的。苯酚的羟基是必需的，因为苯和氟苯是不活跃的。除邻苯二酚外，具有多个羟基的化合物活性不如苯酚本身。高效液相色谱法未检测到细胞与皮质醇孵育过程中产生的苯酚产物。苯酚保护11β-羟基酶的EC50值约为100nM，而二甲基亚砜和甲草隆的EC50值分别为10 mM和300nM。在半剂量曲线上，所有这些化合物的剂量-反应曲线大致平行。为了帮助确定11β-羟基酶的保护机制，在三种不同的氧气浓度(2，5和19%O2)下，测试了酚类和二甲基亚砜对防止11β-羟基酶活性丧失的作用。降低氧气浓度本身会导致11β-羟基酶的损失略有减少。苯酚和二甲基亚砜在低氧条件下效果较好，在空气中效果较差。由于细胞色素P-450抑制剂美泰龙先前被发现在保护11β-羟基酶活性丧失方面非常有效，我们研究了酚类和二甲基亚砜是否可能通过直接抑制11β-羟基酶活性而起作用。在与细胞1小时的孵育中，苯酚和二甲基亚砜都抑制了11β-羟基酶，但在浓度从4-到-gt；100倍的范围内高于保护所需的浓度。相比之下，对于美替拉奉，保护和抑制的EC50值非常相似。这些结果表明，酚不太可能简单地作为抑制剂，但可能需要结合到酶的活性部位附近。观察到的氧与低氧的协同作用表明，11β羟基酶活性的丧失与氧中心自由基有关，氧中心自由基可能是保护性酚的反应物。
When cultured bovine adrenocortical cells are incubated with cortisol, or other steroids that are pseudosubstrates for 11β-hydroxylase (cytochrome P-45011β), the activity of the enzyme decreases. In previous experiments, three substances were shown to protect 11β-hydroxylase against loss of enzymatic activity in the presence of pseudosubstrates: BHA (butylated hydroxyanisole, 2(3)-tert-butyl-4-methoxyphenol), dimethyl sulfoxide (DMSO), and metyrapone. The present experiments examine the protective effects of several phenolic analogs of BHA in this system, and compare their activities to that of DMSO and metyrapone. When a variety of analogs of BHA were tested for their abilities to prevent loss of 11β-hydroxylase activity in cultured adrenocortical cells incubated with 50 μM cortisol for 24 hr, phenol itself was found to be about equipotent with BHA. Addition of methyl, methoxy and benzyl groups to phenol did not diminish protective activity of the compound, but addition of one and particularly twotert-butyl groups greatly diminished activity. Thus, BHT (2,6-di-t-butyl-4-methylphenol) was inactive, in contrast to BHA. The hydroxy group of phenol was essential since benzene and fluorobenzene were inactive. Compounds with multiple hydroxyl groups were not as active as phenol itself, with the exception of catechol. No products of phenol formed during incubations of cells with cortisol were detected by high performance liquid chromatography. Estimatedec50values for protection of 11β-hydroxylase by phenols were about 100 μM, whereas theec50values for dimethyl sulfoxide and metyrapone were 10 mM and 300 nM respectively. On a semilogarothmic plot, the dose-response curves for all these compounds were approximately parallel. To aid in determining the mechanism of protection of 11β-hydroxylase, phenols and DMSO were tested for prevention of loss of 11β-hydroxylase activity at three different oxygen concentrations (2,5, and 19% O2). Lowering the oxygen concentration itself resulted in a small diminution of the loss of 11β-hydroxylase. Phenols and dimethyl sulfoxide were more effective at low oxygen and less effective in air. Because the cytochrome P-450 inhibitor metyrapone was found previously to be very effective in protecting 11β-hydroxylase against loss of activity, we examined whether phenols and dimethyl sulfoxide may act by directly inhibiting 11β-hydroxylase activity. In a 1-hr incubation with cells, BHA, phenol, and dimethyl sulfoxide all inhibited 11β-hydroxylase, but at concentrations that ranged from 4- to >100-fold higher than those required for protection. In contrast, for metyrapone, theec50values for protection and inhibition were very similar. These results indicate that it is unlikely that phenols act simply as inhibitors but may need to bind close to the active site of the enzyme. The observed synergism with lowered oxygen suggests an involvement, in the loss of 11β-hydroxylase activity, of oxygen-centered radicals that may be reactants for protective phenols.
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NADPH依赖性脂质过氧化与肾上腺皮质线粒体细胞色素P-450降解之间的关系。
DOI：
10.1016/0006-291x(83)91186-5
发表时间：
1983
影响因子：
3.1
作者：
Klimek,J;Schaap,AP;Kimura,T
通讯作者：
Kimura,T
类固醇对培养牛肾上腺皮质球状带细胞醛固酮合成的抑制及其抗氧化剂的预防。
影响因子：
4.8
作者：
Crivello,JF;Hornsby,PJ;Gill,GN
通讯作者：
Gill,GN
影响因子：
4.8
作者：
M. H. Simonian;M. White;G. Gill
通讯作者：
M. H. Simonian;M. White;G. Gill
过氧化物氧均裂机制的证据——细胞色素 P-450 底物羟基化过程中氧键断裂。
DOI：
--
发表时间：
1980
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
White,RE;Sligar,SG;Coon,MJ
通讯作者：
Coon,MJ
培养牛肾上腺皮质细胞中类固醇对 21-羟化酶活性的调节：对肾上腺皮质雄激素合成的可能意义。
影响因子：
4.8
作者：
Hornsby,PJ
通讯作者：
Hornsby,PJ