Dick Ryan A, Kanne David B, Casida John E
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley 94720-3112, USA.
Chem Res Toxicol. 2007 Dec;20(12):1942-6. doi: 10.1021/tx700265r. Epub 2007 Nov 15.
The major neonicotinoid insecticide imidacloprid (IMI) is used worldwide for crop protection and pest control on pets. IMI is extensively metabolized, oxidatively by cytochromes P450 and via aerobic nitroreduction by the molybdo-flavoenzyme aldehyde oxidase (AOX). Rabbit liver AOX is capable of reducing IMI to both its nitrosoguanidine (IMI-NO) and aminoguanidine (IMI-NH2) derivatives; however, when IMI-NO is used as a substrate, less than stoichiometric amounts of IMI-NH2 are detected while IMI-NO is completely consumed. The disappearance of IMI-NO requires both a source of AOX and an AOX-specific electron donor substrate and is not inhibited by the addition of catalase and superoxide dismutase. Experiments to evaluate IMI-NO as a possible time-dependent inactivator of AOX reveal the following four characteristics: First, partially purified AOX (ppAOX) is inactivated at a moderate rate by the electron donor substrate N-methylnicotinamide (NMN); second, AOX is inactivated by IMI-NO in an NMN-dependent manner at a 10-fold greater rate; third, IMI does not inactivate AOX; and finally, GSH protects AOX from inactivation but not to a degree greater than IMI-NO-deficient incubations. Values for the kinetic constants of KI and kinact are measured to be 1.3 mM and 0.35 min(-1), respectively. Ultrafiltration is used to establish that IMI-NO inactivation is not reversible and to determine a partition ratio of 1.6. [3H]IMI-NO labeling shows that significant amounts (19%) of this molecule covalently bind to protein following reduction by ppAOX. The addition of 10 mM GSH attenuates this binding almost completely. These findings demonstrate that IMI-NO is metabolically activated by rabbit AOX to form both an irreversible inhibitor and a reactive intermediate that is capable of covalently binding to protein.
主要的新烟碱类杀虫剂吡虫啉（IMI）在全球范围内用于作物保护和宠物害虫防治。IMI会被广泛代谢，通过细胞色素P450进行氧化代谢，并通过钼黄素酶醛氧化酶（AOX）进行需氧硝基还原代谢。兔肝AOX能够将IMI还原为其亚硝基胍（IMI-NO）和氨基胍（IMI-NH2）衍生物；然而，当使用IMI-NO作为底物时，在IMI-NO完全消耗的情况下，检测到的IMI-NH2量少于化学计量。IMI-NO的消失既需要AOX来源，也需要AOX特异性电子供体底物，并且不受过氧化氢酶和超氧化物歧化酶添加的抑制。评估IMI-NO作为AOX可能的时间依赖性失活剂的实验揭示了以下四个特征：第一，部分纯化的AOX（ppAOX）被电子供体底物N-甲基烟酰胺（NMN）以中等速率失活；第二，AOX被IMI-NO以NMN依赖性方式失活，失活速率快10倍；第三，IMI不会使AOX失活；最后，谷胱甘肽（GSH）可保护AOX免于失活，但保护程度不超过缺乏IMI-NO的孵育。测得的抑制常数（KI）和失活速率常数（kinact）值分别为1.3 mM和0.35 min-1。使用超滤来确定IMI-NO失活是不可逆的，并确定分配比为1.
