Hamers T, Molin K R, Koeman J H, Murk A J
Toxicology Group, Wageningen University, P.O. Box 8000, 6700 EA Wageningen, The Netherlands.
Toxicol Sci. 2000 Nov;58(1):60-7. doi: 10.1093/toxsci/58.1.60.
The goal of this study was to develop a sensitive in vitro bioassay for quantification of the total esterase inhibiting potency of low concentrations of organophosphate and carbamate insecticides in relatively small rainwater samples. Purified acetylcholinesterase (AChE) from electric eel (Electrophorus electricus) and carboxylesterases from a homogenate of honeybee heads (Apis mellifera) were used as esterases, each having different affinities for the substrates S-acetylthiocholine-iodide (ATC) and N-methylindoxylacetate (MIA). MIA hydrolysis by honeybee homogenate was more sensitive to inhibition by organophosphate insecticides than ATC hydrolysis by purified AChE, although the latter parameter is often used for in vitro monitoring of esterase inhibitors. The higher sensitivity of carboxylesterases is attributed to the instant formation of a reversible Michaelis-Menten complex with the inhibitor, which competes with MIA for the active sites of the free enzymes. This dose-dependent instant inhibition can be quantified with kinetics for competitive inhibition at dichlorvos concentrations < 16 nM. At similar concentrations, purified AChE was not instantly inhibited, whereas both AChE and carboxylesterases were irreversibly and progressively inhibited at higher dichlorvos concentrations (IC50(10min) >/= 0.1 microM). Honeybee homogenate mediated MIA hydrolysis was applied as the most sensitive enzyme-substrate combination for experiments with fractionated extracts of 4 rainwater samples collected in a natural conservation area. Most esterase inhibiting potency was found in the polar methanol fraction, with recalculated concentrations equivalent to 12-125 ng dichlorvos per liter rainwater.
本研究的目的是开发一种灵敏的体外生物测定法，用于定量分析相对少量雨水样本中低浓度有机磷和氨基甲酸酯类杀虫剂的总酯酶抑制效力。用电鳗（Electrophorus electricus）纯化的乙酰胆碱酯酶（AChE）和蜜蜂头部匀浆（Apis mellifera）中的羧酸酯酶作为酯酶，它们对底物硫代乙酰胆碱碘化物（ATC）和N-甲基吲哚乙酸酯（MIA）具有不同的亲和力。尽管后一参数常用于体外监测酯酶抑制剂，但蜜蜂匀浆对MIA的水解比纯化的AChE对ATC的水解对有机磷杀虫剂的抑制更敏感。羧酸酯酶较高的敏感性归因于与抑制剂瞬间形成可逆的米氏复合物，该复合物与MIA竞争游离酶的活性位点。在敌敌畏浓度<16 nM时，这种剂量依赖性的瞬间抑制可用竞争性抑制动力学进行定量。在相似浓度下，纯化的AChE不会被瞬间抑制，而在较高敌敌畏浓度（IC50(10min)≥0.1 microM）时，AChE和羧酸酯酶都会被不可逆地逐步抑制。蜜蜂匀浆介导的MIA水解作为最灵敏的酶-底物组合，用于对在一个自然保护区采集的4个雨水样本的分级提取物进行实验。大多数酯酶抑制效力存在于极性甲醇馏分中，重新计算后的浓度相当于每升雨水12-125 ng敌敌畏。
