Santillo Michael F, Liu Yitong
Division of Toxicology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD 20708, United States.
Division of Toxicology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD 20708, United States.
J Pharmacol Toxicol Methods. 2015 Nov-Dec;76:15-22. doi: 10.1016/j.vascn.2015.07.002. Epub 2015 Jul 9.
Acetylcholinesterase (AChE) is an enzyme responsible for metabolism of the neurotransmitter acetylcholine, and inhibition of AChE can have therapeutic applications (e.g., drugs for Alzheimer's disease) or neurotoxic consequences (e.g., pesticides). A common absorbance-based AChE activity assay that uses 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) can have limited sensitivity and be prone to interference. Therefore, an alternative assay was developed, in which AChE activity was determined by measuring fluorescence of resorufin produced from coupled enzyme reactions involving acetylcholine and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). The Amplex Red assay was used for two separate applications. First, AChE activity was measured in rat whole blood, which is a biomarker for exposure to AChE inhibitor pesticides. Activity was quantified from a 10(5)-fold dilution of whole blood, and there was a linear correlation between Amplex Red and DTNB assays. For the second application, Amplex Red assay was used to measure AChE inhibition potency in a human neuroblastoma cell line (SH-SY5Y), which is important for assessing pharmacological and toxicological potential of AChE inhibitors including drugs, phytochemicals, and pesticides. Five known reversible inhibitors were evaluated (IC50, 7-225 nM), along with irreversible inhibitors chlorpyrifos-oxon (ki=1.01 nM(-1)h(-1)) and paraoxon (ki=0.16 nM(-1)h(-1)). Lastly, in addition to inhibition, AChE reactivation was measured in SH-SY5Y cells incubated with pralidoxime chloride (2-PAM). The Amplex Red assay is a sensitive, specific, and reliable fluorescence method for measuring AChE activity in both rat whole blood and cultured SH-SY5Y cells.
乙酰胆碱酯酶（AChE）是一种负责神经递质乙酰胆碱代谢的酶，抑制AChE可产生治疗作用（如用于治疗阿尔茨海默病的药物）或神经毒性后果（如杀虫剂）。一种常用的基于吸光度的AChE活性测定方法，使用5,5'-二硫代双（2-硝基苯甲酸）（DTNB），其灵敏度可能有限且容易受到干扰。因此，开发了一种替代测定方法，其中通过测量由涉及乙酰胆碱和Amplex Red（10-乙酰基-3,7-二羟基吩恶嗪）的偶联酶反应产生的试卤灵荧光来测定AChE活性。Amplex Red测定法用于两个不同的应用。首先，在大鼠全血中测量AChE活性，大鼠全血是接触AChE抑制剂农药的生物标志物。从全血10⁵倍稀释液中定量活性，Amplex Red测定法与DTNB测定法之间存在线性相关性。对于第二个应用，Amplex Red测定法用于测量人神经母细胞瘤细胞系（SH-SY5Y）中的AChE抑制效力，这对于评估包括药物、植物化学物质和杀虫剂在内的AChE抑制剂的药理和毒理潜力很重要。评估了五种已知的可逆抑制剂（IC50，7-225 nM），以及不可逆抑制剂毒死蜱氧磷（ki = 1.01 nM⁻¹h⁻¹）和对氧磷（ki = 0.16 nM⁻¹h⁻¹）。最后，除了抑制作用外，还在与氯解磷定（2-PAM）孵育的SH-SY5Y细胞中测量了AChE的重新激活。Amplex Red测定法是一种灵敏、特异且可靠的荧光方法，用于测量大鼠全血和培养的SH-SY5Y细胞中的AChE活性。
