State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226-0509, USA.
Free Radic Biol Med. 2019 Jan;130:1-7. doi: 10.1016/j.freeradbiomed.2018.10.425. Epub 2018 Oct 21.
We have recently shown that the pyridinium aldoximes, best-known as therapeutic antidotes for chemical warfare nerve-agents, could markedly detoxify the carcinogenic tetrachloro-1,4-benzoquinone (TCBQ) via an unusual double Beckmann fragmentation mechanism. However, it is still not clear why pralidoxime (2-PAM) cannot provide full protection against TCBQ-induced biological damages even when 2-PAM was in excess. Here we show, unexpectedly, that TCBQ can also activate pralidoxime to generate a reactive iminyl radical intermediate in two-consecutive steps, which was detected and unequivocally characterized by the complementary application of ESR spin-trapping, HPLC/MS and nitrogen-15 isotope-labeling studies. The same iminyl radical was observed when TCBQ was substituted by other halogenated quinones. The end product of iminyl radical was isolated and identified as its corresponding reactive and toxic aldehyde. Based on these data, we proposed that the reaction of 2-PAM and TCBQ might be through the following two competing pathways: a nucleophilic attack of 2-PAM on TCBQ forms an unstable transient intermediate, which can decompose not only heterolytically to form 2-CMP via double Beckmann fragmentation, but also homolytically leading to the formation of a reactive iminyl radical in double-steps, which then via H abstraction and further hydrolyzation to form its corresponding more toxic aldehyde. Analogous radical homolysis mechanism was observed with other halogenated quinones and pyridinium aldoximes. This study represents the first detection and identification of reactive iminyl radical intermediates produced under normal physiological conditions, which provides direct experimental evidence to explain only the partial protection by 2-PAM against TCBQ-induced biological damages, and also the potential side-toxic effects induced by 2-PAM and other pyridinium aldoxime nerve-agent antidotes.
我们最近表明，吡啶醛肟作为化学战神经毒剂的治疗解毒剂，通过一种不寻常的双贝克曼重排机制，可以显著解毒致癌的四氯-1,4-苯醌（TCBQ）。然而，目前尚不清楚为什么即使普洛肟（2-PAM）过量，它也不能为 TCBQ 诱导的生物损伤提供完全的保护。在这里，我们出人意料地表明，TCBQ 还可以在两个连续步骤中激活普洛肟生成反应性亚氨自由基中间体，这通过 ESR 自旋捕获、HPLC/MS 和氮-15 同位素标记研究的互补应用得到了检测和明确的表征。当 TCBQ 被其他卤代醌取代时，也观察到相同的亚氨自由基。亚氨自由基的终产物被分离并鉴定为其相应的反应性和毒性醛。基于这些数据，我们提出 2-PAM 和 TCBQ 的反应可能通过以下两种竞争途径进行：2-PAM 的亲核攻击 TCBQ 形成不稳定的瞬态中间体，它不仅可以通过双贝克曼重排异裂分解，而且可以通过两步均裂形成反应性亚氨自由基，然后通过 H 提取和进一步水解形成其相应的更毒性醛。类似的自由基均裂机制也在其他卤代醌和吡啶醛肟中观察到。本研究首次检测和鉴定了在正常生理条件下产生的反应性亚氨自由基中间体，为仅部分保护 2-PAM 免受 TCBQ 诱导的生物损伤提供了直接的实验证据，也为 2-PAM 和其他吡啶醛肟神经毒剂解毒剂引起的潜在副作用提供了直接的实验证据。
