A fluorescence assay for measuring acetylcholinesterase activity in rat blood and a human neuroblastoma cell line (SH-SY5Y)

• Published in: Journal of pharmacological and toxicological methods 2015-11, Vol.76, p.15-22
• Main Authors: Santillo, Michael F., Liu, Yitong
• Format: Article
• Language: English
• Subjects: 10-Acetyl-3,7-dihydroxyphenoxazine; Acetylcholine - metabolism; Acetylcholinesterase (AChE); Acetylcholinesterase - blood; Acetylcholinesterase - metabolism; Amplex Red; Animals; Blood; Cell assay; Cell Culture Techniques; Cell Line, Tumor; Cholinesterase Inhibitors - analysis; Enzyme inhibitors; Enzyme kinetics; Fluorescence; Fluorometry; Humans; In vitro; Neurons - enzymology; Neurotoxicity; Oxazines - analysis; Oxazines - chemistry; Rats, Wistar; Sensitivity and Specificity; Substrate Specificity
• ISSN: 1056-8719; 1873-488X
• DOI: 10.1016/j.vascn.2015.07.002

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 105-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, 7–225nM), along with irreversible inhibitors chlorpyrifos-oxon (ki=1.01nM−1h−1) and paraoxon (ki=0.16nM−1h−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. [Display omitted]