Comparative Responses Of Acetylcholinesterase (Ache) Of  Rat Brain And Model Invertebrate Caenorhabditis Elegans In Vitro

Kisan B Jadhav; Shashikumar Shivaya; Hari Prasad; Apurva Kumar Joshi; Revanappa Biradar; B S Janagoudar; P S Rajini

doi:10.48165/

Authors

Kisan B Jadhav Main Agricultural Research Station, Molecular Biology Unit, University of Agricultural Sciences, Raichur, Karnataka (India)
Shashikumar Shivaya Food Protectants and Infestation Control Department, Central Food Technological Research Institute (CSIR Lab), Chaluvamba Mansion, Mysore 570 020, Karnataka (India)
Hari Prasad Food Protectants and Infestation Control Department, Central Food Technological Research Institute (CSIR Lab), Chaluvamba Mansion, Mysore 570 020, Karnataka (India)
Apurva Kumar Joshi Food Protectants and Infestation Control Department, Central Food Technological Research Institute (CSIR Lab), Chaluvamba Mansion, Mysore 570 020, Karnataka (India)
Revanappa Biradar Main Agricultural Research Station, Molecular Biology Unit, University of Agricultural Sciences, Raichur, Karnataka (India)
B S Janagoudar Main Agricultural Research Station, Molecular Biology Unit, University of Agricultural Sciences, Raichur, Karnataka (India)
P S Rajini Food Protectants and Infestation Control Department, Central Food Technological Research Institute (CSIR Lab), Chaluvamba Mansion, Mysore 570 020, Karnataka (India)

DOI:

https://doi.org/10.48165/

Keywords:

Acetylcholine, acetylcholinesterase, Caenorhabditis elegans, organophosphorus insecticides, rat brain homogenate

Abstract

This in vitro study was aimed to compare the kinetic properties of acetyl cholinesterase (AChE) in two model systems i.e. rat brain homogenate and nematode model Caenorhabditis elegans. These models were analyzed with basic kinetic parameters viz., substrate hydrolysis, inhibition pattern, determined Km, Vmax, IC50, KI and Vimax using dichlorvos (DDVP), an organophosphorus compound widely used for insect-pest control in agricultural crops. The rat brain homogenate exhibited Km, Vmax and IC50 values of 59 M, 33 nmol min-1 mg-1protein and 107.18 ± 3.56 nM, respectively, for rat brain homogenate. C. elegans homogenate exhibited Km, Vmax and IC50 values of 725.41 M, 112.35 nmol min-1 mg-1protein and 45.7 ± 2.68 nM, respectively. The pattern of inhibition was irreversible in both the systems. The study showed that C. elegans in vitro system can be used as a model to establish the inhibition pattern of organophosphorus compounds (known inhibitors of AChE) as compared to traditionally used rat brain homogenate system.

Downloads

Download data is not yet available.

References

Ali, H.M., Attia, M.H. and Mohamed, K.A. 2003. Biodisposition and biochemical effects of a new phosphoramidate series in rat tissues. Bulletin of Environmental Contamination and Toxicology, 70: 1197-1204.

Anderson, G.L. 1978. Responses of dauer larvae of Caenorhabditis elegans (Nematoda: Rhabd itidae) to thermal stress and oxygen deprivation. Canadian Journal Zoology, 56: 1786-1791.

Acetylcholinesteratse of rat brain and Caenorhabditis elegans 175

Brenner, S. 1974. The genetics of Caenorhabditis elegans. Genetics 77: 71-94. Combes, D., Fedon, Y. and Toutant, J. 2001. Acetylcholinesterase genes in the nematode Caenorhabditis elegans. International Review on Cytology, 209: 207-239.

Ellman, G.L., K.D., Courtney, V., Andres J.R. and Featherstone, R.M. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem.Pharmacology, 7: 88-95. Filho, M.V., Oliveira, M.M., Salles, J.B., Bastos, V.L.F.C., Cassino, V.P.F. and Bastos, J.C. 2004. Methyl paraxon comparative inhibition kinetics for acetylcholinesterase from brain of neotropical fishes. Toxicology Letters, 153: 247-254.

Franz, W., Peter E. and Horst T. 2012. Determination of acetlycholinesterases activity by the Ellman assay: A versatile tool for in vitro research on medical counter measures against organophosphate poisoning. Drug Testing and Analysis, 4: 282-291.

Gagnaire, B., Geffard, O., Xuereb, B., Margoum, C. and Garric, J. 2008. Cholinesterase activities as potential biomarkers: Characterization in two freshwater snails, Potamopyrgus antipodarum (Mollusca, Hydrobiidae, Smith 1889) and Valvata piscinalis (Mollusca, Valvatidae, Müller 1774). Chemosphere, 71: 553-560.

Jadhav, K.B. and Rajini P.S. 2009a. Neurophysiological alterations in C. elegans exposed to dichlorvos, an OPI. Pesticide Biochemistry and Physiology, 94: 79-85.

Jadhav, K.B. and Rajini, P.S. 2009b. Evaluation of sublethal effects of dichlorvos upon Caenorhabditis elegans based on a set of end points of toxicity. Journal of Biochemistry and Molecular Toxicology, 23: 9-17.

Marcel, V., Gagnoux, P.L., Pertuy, C.M. and Fournier, D. 1998. Two invertebrate acetylcholin esterases show activation followed by inhibition with substrate concentration. Biochemistry Journal, 329: 329-334.

Nonet, M.L., Saifee, O., Zhao, H., Rand, J.B. and Wei, L. 1998. Synaptic transmission deficits in Caenorhabditis elegans synaptobrevin mutants. Journal of Neurosciences, 18: 70-80. Pohahanka, M., Hrabinova, M., Kamila K. and Jean-Pierre Simanato. 2011. Assessment of acetyl cholinesterase activity using indolacetate and comparison with the standard Ellman’s method. International Journal of Molecular Sciences, 12: 2631-2640.

Rajini, P.S., Melstrom, P.C. and Williams, P.L. 2008. A comparative study on the relationship between various toxicological endpoints in Caenorhabditis elegans exposed to organophosphorus insecticides. Toxicology Environment Health, 71: 1043-1050. Rand, J.B. 2007. Acetylcholine. Wormbook. http://www.wormbook.org.

Rand, J.B. and Nonet, M.L. 1997 Synaptic transmission. pp. 611-643. In: C. elegans II (eds. D.L. Riddle, T. Blumenthal, B.J. Meyer and J.R. Priess). Cold Spring Harbor Press, NewYork, USA. Reiner, E., Skrinjaric-spoljar, M., Kralg, M. and Krvavica, S. 1978. Kinetic properties of the cholinesterase in Metastrongylus apri (Nematoda): Substrate hydrolysis and reaction with

organophosphorus compounds. Compound Biochemistry and Physiology, 60C: 155-157. Rhoads, M.L. 1981. Cholinesterase in the parasitic nematrode, Stephanurus dentatus. Journal of Biological Chemistry, 256: 9316 - 9323.

Sanderson, R.T. 1967. Inorganic Chemistry. Rhinhold Publishing Co., New York, USA. Singh, A.K. 1985. Kinetic analysis of inhibition of brain and red blood cellacetyl-cholinesterase and plasma cholinesterase by acephate or methamidophos. Toxicology and Application of Pharmacology, 81: 302-309.

Wang, C. and Murphy, S.D. 1982. Kinetic analysis of species difference in acetylcholinesterase sensitivity to organophosphate insecticides. Toxicology and Applied Pharmacology, 66: 409- 419.

Williams, P.L. and Dusenbery, D.B. 1990. Aquatic toxicity testing using the nematode, Caenorhabditis elegans. Environmental Toxicology Chemistry, 9: 1285-1290. Zhu, K.Y. and Clark, J.M. 1994. Purification and characterization of acetylcholinesterase from the Colarado potato beetle Leptinotarsa decemlinelata (Say). Insect Biochemistry and Molecular Biology, 24: 453-461.