Jokanovic M, Johnson M K
MRC Toxicology Unit, MRC Laboratories, Carshalton, Surrey, United Kingdom.
J Biochem Toxicol. 1993 Mar;8(1):19-31. doi: 10.1002/jbt.2570080105.
For organophosphates or phosphonates to initiate delayed neuropathy two steps are necessary: (1) progressive covalent reaction with neuropathy target esterase (NTE) to produce a form of inhibited NTE which can be reactivated by incubation with aqueous potassium fluoride (KF) and (2) progressive "aging" of inhibited NTE to a form which can no longer be reactivated by KF. However, it has been shown recently that certain N-unsubstituted organophosphoro-monoamidates (analogues of methamidophos) cause delayed neuropathy even though the inhibited NTE appeared not to have aged (Johnson et al. (1991). Arch. Toxicol., 65, 618-624). In order to study the generality of this phenomenon, we have examined some N-substituted compounds. We report in vitro studies of inhibition and reactivation and aging of both NTE and acetylcholinesterase (AChE) prior to toxicological tests. All the compounds studied were less inhibitory to both NTE and AChE in concentrated rather than in dilute suspensions of EDTA-washed brain particles without added cofactors. There was an apparent disposal of up to 100 mumoles of test compound by particles from 95 mg hen brain, which is far greater than can be explained by covalent binding. The activity is distinct from calcium-dependent "A" esterase. Several N-alkyl phosphoromonoamidates were found to be potent and selective inhibitors of NTE: second-order rate constant for O-n-pentyl N-benzylphosphoramido-fluoridate (Cmpd 6) = 5.6 x 10(7) M-1 min-1 at 37 degrees, which is about 100x higher than for acetylcholinesterase (AChE). Inhibited NTE and AChE from several chiral phosphoromono-amidates did not reactivate spontaneously (21 hours at 37 degrees). Virtually 100% reactivation by KF of AChE inhibited by phosphoromonoamidates was achieved at all times tested. Acetylcholinesterase inhibited by 2,5-dichlorophenyl N,N'-di-n-butylphosphorodiamidate was 42-56% reactivated by incubation with KF (192 mM in pH 5.2 buffer for 30 minutes at 37 degrees). We believe this is the first report of reactivation of any enzyme after inhibition by a phosphorodiamidate. For NTE inhibited by tabun (O-ethyl N-dimethylphosphoroamidocyanidate), virtually complete and rapid aging (t1/2 = 5.5-8.4 minutes) was observed. Consistent but only partial reactivation by KF was achieved 2 or more hours after inhibition of NTE by Cmpd 6 or by its 2,6-difluoro-analogue (Cmpd 7). However, a small but significant aging (approximately 15-20% loss of reactivatability) was measured soon after a 1 minute inhibition by Cmpd 7, but no further change occurred in 21 hours.(ABSTRACT TRUNCATED AT 400 WORDS)
（1）与神经病靶酯酶（NTE）进行渐进性共价反应，生成一种可通过与氟化钾（KF）水溶液孵育而重新活化的抑制形式的NTE；（2）抑制形式的NTE进行渐进性“老化”，转变为一种不能再被KF重新活化的形式。然而，最近研究表明，某些N-未取代的有机磷单酰胺（甲胺磷类似物）会引发迟发性神经病，尽管被抑制的NTE似乎并未老化（约翰逊等人，《毒理学文献》，1991年，第65卷，第618 - 624页）。为了研究这一现象的普遍性，我们研究了一些N-取代化合物。我们报告了在毒理学测试之前，对NTE和乙酰胆碱酯酶（AChE）的抑制、重新活化及老化的体外研究。在没有添加辅助因子的情况下，所有研究的化合物在EDTA洗涤的脑颗粒浓缩悬浮液中对NTE和AChE的抑制作用均小于稀释悬浮液。95毫克鸡脑颗粒对高达100微摩尔的测试化合物有明显的清除作用，这远远超过共价结合所能解释的量。该活性不同于钙依赖性“A”酯酶。发现几种N-烷基磷单酰胺是NTE的有效且选择性抑制剂：O-正戊基N-苄基磷酰胺氟化物（化合物6）在37℃时的二级速率常数 = 5.6×10⁷ M⁻¹ min⁻¹，这比乙酰胆碱酯酶（AChE）高约100倍。几种手性磷单酰胺抑制后的NTE和AChE不会自发重新活化（37℃下21小时）。在所有测试时间，由磷单酰胺抑制的AChE几乎都能被KF完全重新活化。2,5-二氯苯基N,N'-二正丁基磷二酰胺抑制的乙酰胆碱酯酶，通过在37℃下于pH 5.2缓冲液中与192 mM KF孵育30分钟，可重新活化42 - 56%。我们认为这是关于磷二酰胺抑制后任何酶重新活化的首次报道。对于被塔崩（O-乙基N-二甲基磷酰胺氰化物）抑制的NTE，观察到几乎完全且快速的老化（半衰期 = 5.5 - 8.4分钟）。在化合物6或其2,6-二氟类似物（化合物7）抑制NTE 2小时或更长时间后，KF能实现一致但仅部分的重新活化。然而，在化合物7抑制1分钟后不久，检测到轻微但显著的老化（重新活化能力约损失15 - 20%），但在21小时内没有进一步变化。（摘要截取自400字）
