The use of selective inhibitors to estimation of cholinesterases activity of liver and muscle for food animals

Kasim Abass Askar, Caleb Kudi, John Moody. School of Biomedical and Biological Sciences, Faculty of Science and Technology, University of Plymouth, Plymouth, PL4 8AA, United Kingdom.

Background: Cholinesterases (ChE) are a pivotal enzyme in the cholinergic nervous system. Its primary function is to catalyze hydrolysis of released acetylcholine and thus maintain homeostasis of this neurotransmitter in the central and peripheral nervous systems. They are distinguished according to their sensitivity to the selective inhibitors as acetylcholinesterase (AChE) or butyrylcholinesterase (BChE). The purpose of this study was to investigate the presence of different ChE by using two selective inhibitors for AChE and BChE. A further aim was to study the AChE as well as the presence of BChE in liver and muscles of food animals (sheep, cattle, and pigs). To this end of study, determine the values of enzymes activity as biomarkers of pesticides compounds exposure and increases awareness in worker to pesticides compounds.

Materials and Methods: Meat from food animals (n = 5-10) was obtained from local abattoirs and transported in a cool box to the laboratory. To extract ChE, one gram of each tissue (Liver and Muscle) were removed using a scalpel, cut into small pieces (3-5 mm3), and homogenized with sodium phosphate buffer (0.1 M, pH 8) at a ratio of 1:9 of buffer. The homogenate was then centrifuged at 9000 g for 5 min at 4°C. ChE activity was determined by (1), adapted for use with microtitre plates as described by (2), and using; acetylthiocholine iodide (ATCI), butyrylthiocholine iodide (BTCI), or propionylthiocholine iodide (PTCI) as substrate (1 mM final concentration) for measuring AChE and BChE activities, respectively. Briefly, 0.02 ml of sample and 0.24 ml of assay mixture [9.75 ml of 0.1 M sodium phosphate buffer, pH 8.0, containing 1 mM EDTA, and 0.25 ml of 0.2 mM 5,5’-dithiobis-(2-nitrobenzoic acid)] were mixed, allowed to stand for 5 min, and then 0.04 ml of substrate solution were added. The absorbance was monitored for 5 min at 410 nm, at 25°C in a plate reader (OptiMax, Molecular Devices, Sunnyvale, CA) (2). The inhibitors 1: 5-bis (4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51) and tetraisopropyl pyrophosphoramide (iso-OMPA) (identified in mammalian studies as diagnostic inhibitors of AChE and BChE, respectively). Final inhibitor concentrations ranging between 0.5 and 16 × 10-3 M for iso-OMPA and from 0.25 to 8 × 10-6 M for BW284c5 (3).

Results: Inhibition effect for liver in animal to BW284c51 are increased according to rank order of pig > cattle > sheep for ATCI and PTCI while it increased: cattle > sheep > pig for BTCI. Liver iso-OMPA increases as follows: cattle > pig > sheep for ATCI and cattle > sheep > pig for BTCI, while increased pig > cattle > sheep for PTCI. Liver percentage inhibition of BW284c51 was almost ranged ATCI (40.7–94%), BTCI (8.5-79.7%), and PTCI (50.2-80.7%), while iso-OMPA ranged ATCI (23.8-75.3%), BTCI (41.1-63.8%), and PTCI (13.9-48.2%). Muscle rates inhibition in animals to BW284c51 are increased as follows: pig > sheep > cattle for ATCI and PTCI, while it increased: cattle > pig > sheep for BTCI. Muscle iso-OMPA in animals increased as follows: pig > cattle > sheep for ATCI and pig > cattle > sheep for BTCI, while increased cattle > pig > sheep for PTCI. Percentage muscle inhibition of BW284c51 ranged ATCI (51.9-81.5%), BTCI (9.3-30%), and PTCI (32.7-87.5%), whereas iso-OMPA for ATCI (21.6-27.2%), BTCI (10.5-29.5%), and PTCI (18-65.8%). Pearson correlation coefficient (r) calculated to measure the degree of relationship between iso-OMPA and BW284c51 in the liver and muscles for tested animals was observed significant (ANOVA, P < 0.05 and r > 0.84), with the exception of muscle where BTCI activity was poor correlation and insignificant (ANOVA, P = 0.087, r = 0.69) for sheep and (ANOVA, P = 0.137, r = 0.62) for cattle.

Conclusions: ATCI indicate that, preferential substrate for detect ChE selective inhibition of muscle and liver. In general in all three animals species (sheep, cattle, and pigs) the liver and muscle tissues were most sensitive to inhibition with BW284c51 than iso-OMPA by all three substrates used (ATCI, BTCI, and PTCI), with exception for BTCI in sheep and pig cattle iso-OMPA was higher than BW284c51the in liver. As well as, in muscle BTCI for sheep and PTCI for cattle were stronger inhibition in iso-OMPA than BW284c51.

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