Toxic effects of diazinon and its degradation products, diazoxon and 2- isopropyl-6-methyl-4-pyrimidinol (IMP), were investigated in vitro by determining the inhibition of acetylcholinesterase (AChE), Na+ /K+ -ATPase and ecto-ATPase activity in rat brain synaptosomes after 1 hour exposure toward varying concentrations. Dose-dependent AChE and Na+ /K+ -ATPase inhibition was obtained in the presence of diazinon, while diazinon concentrations below 0.1 mM did not noticeably affect ecto-ATPase activity. Diazinon oxidation product, diazoxon was found as the most toxic investigated compound. Diazoxon induced dose-dependent and almost complete inhibition of AChE, Na+ /K+ -ATPase and ecto-ATPase at the highest investigated concentration (0.1 mM), while hydrolysis product of diazinon, IMP did not remarkably influence their activities

Avramović, Nataša

Krstić, Danijela Z.

Vasić, Vesna M.

Čolović, Mirjana B.

Đurić, Dragan M.

Repository of the Vinča Nuclear Institute (VinaR)

     PHYSICAL CHEMISTRY 2014  12th International Conference on Fundamental and Applied Aspects of Physical Chemistry          The Conference is dedicated to the 25. Anniversary of the Society of Physical Chemists of Serbia            September 22-26, 2014  Belgrade, Serbia   II   ISBN 978-86-82475-30-9 Title: PHYSICAL CHEMISTRY 2014 (Proceedings) Editors: Ž. Čupić and S. Anić Published by: Society of Physical Chemists of Serbia, Studenski trg 12-16, 11158, Belgrade, Serbia Publisher: Society of Physical Chemists of Serbia For Publisher: S. Anić, President of Society of Physical Chemists of Serbia Printed by: “Jovan” Priting and Publishing Company; 200 Copies; Number of pages: 6+ 441; Format: B5; Printing finished in September 2014.  Text an Layout: “Jovan”  Neither this book nor any part may be reproduced or transmitted in any form or by any means, including photocopying, or by any information storage and retrieval system, without permission in writing from the publisher.  200 - Coppy priting    IV  PHYSICAL CHEMISTRY 2014  12th International Conference on  Fundamental and Applied Aspects of Physical Chemistry     Organized by The Society of Physical Chemists of Serbia  in co-operation with   Institute of Catalysis Bulgarian Academy of Sciences   Boreskov Institute of Catalysis of Siberian Branch of the Russian Academy of Sciences  Faculty of Physical Chemistry, University of Belgrade, Serbia  Institute of Chemistry Technology and Metallurgy,  University of Belgrade, Serbia  Vinča Institute, University of Belgrade, Serbia  Institute of General and Physical Chemistry, Serbia    Faculty of Pharmacy, University of Belgrade, Serbia    VInternational Organizing Committee  Chairman:  S. Anić (Serbia) Vice-chairman:  M. Gabrovska (Bulgaria)                             V. A. Sadykov  (Russia)  Members: N. Cvjetičanin (Serbia), S. N. Blagojević (Serbia), M. Daković (Serbia), T. Grozdić (Serbia), D. Jovanović (Serbia), M. Kuzmanović (Serbia), D. Marković (Serbia), J. Marković-Dimitrić (Serbia), B. Milosavljević (USA), M. Mojović (Serbia), N. Ostrovski (Serbia), I. Pašti (Serbia), M. Petković (Serbia), A. Popović-Bjelić (Serbia),  B. Simonović (Serbia), D. Stanisavljev (Serbia),  B. Šljukić (Serbia), N. Vukelić (Serbia), V. Vukojević (Sweden)  International Scientific Committee  Chairman:  Ž. Čupić (Serbia) Vice-chairmans: V. N. Parmon (Russia)                                   S. Rakovsky (Bulgaria) Members: B. Adnađević (Serbia), S. Anić (Serbia), A. Antić-Jovanović (Serbia), G. Bačić (Serbia), A. Kelarakis (Greece), R. Cervellati (Italy), V. Dondur (Serbia), Ivan Gutman (Serbia), S. D. Furrow (USA), K. Hedrih (Serbia), M. Jeremić (Serbia), A. V. Knyazev (Russia), Lj. Kolar-Anić (Serbia), V. Kuntić (Serbia), Z. Marković (Serbia),  S. Mentus (Serbia), Š. Miljanić (Serbia), M. Perić (Serbia), M. Plavšić (Serbia), D. M. F. Santos (Portugal), G. Schmitz (Belgium), I. Schreiber (Czech), P. Sevčik (Slovakia), B. C. Simionescu (Romania), N. Stepanov (Russia), D. Todorović (Serbia), M. Trtica (Serbia), V. Vasić (Serbia), D. Veselinović (Serbia)   Local Executive Committee  Chairman:              S. Blagojević Vice-chairmans:    A. Ivanović-Šašić  Members: P. Banković,  N. Bošnjaković, J. Dostanić,  A. Đerić, A. Ignjatović, A. Jović, N. Jović-Jovičić, D. Lončarević, J. Krstić, J. Maksimović, V. Marković, M. Milenković, S. Maćešić, V. Marković, B. Nedić, N. Potkonjak, D. Ranković, M. Stević, M. Žunić, M. Ristić,          PHYSICAL CHEMISTRY 2014  F-05-P   461IN VITRO EVALUATION OF DIAZINON AND ITS DEGRADATION PRODUCTS NEUROTOXICITY POTENTIAL IN RAT BRAIN SYNAPTOSOMES   M. Čolović1, V. Vasić1, N. Avramović2, D.Djurić3 and D.Krstić2  1 Department of Physical Chemistry, Vinča Institute of Nuclear Sciences, University of Belgrade, Serbia 2Institute of Medical Chemistry, School of Medicine, University of Belgrade, Serbia 3Institute of Medical Physiology “Richard Burian”, School of Medicine, University of Belgrade, Serbia  ABSTRACT Toxic effects of diazinon and its degradation products, diazoxon and 2-isopropyl-6-methyl-4-pyrimidinol (IMP), were investigated in vitro by determining the inhibition of acetylcholinesterase (AChE), Na+/K+-ATPase and ecto-ATPase activity in rat brain synaptosomes after 1 hour exposure toward varying concentrations. Dose-dependent AChE and Na+/K+-ATPase inhibition was obtained in the presence of diazinon, while diazinon concentrations below 0.1 mM did not noticeably affect ecto-ATPase activity. Diazinon oxidation product, diazoxon was found as the most toxic investigated compound. Diazoxon induced dose-dependent and almost complete inhibition of AChE, Na+/K+-ATPase and ecto-ATPase at the highest investigated concentration (0.1 mM), while hydrolysis product of diazinon, IMP did not remarkably influence their activities.   INTRODUCTION Organophosphorus pesticides (OPs) are the most widely used pesticides worldwide and their metabolites are widespread across different populations. The primary mechanism of OP toxicity is the inhibition of AChE in the central and peripheral nervous system [1]. Diazinon (O,O-diethyl-O-(2-isopropyl-4-methyl-6-pyrimidinyl phosphorothionate) is a commonly used thionophosphorous OP to control a variety of insects in agriculture and household environment [2]. Diazinon can be transformed to the more toxic diazoxon due to the enzymatic reaction in birds, fish, insects and mammals [3], while IMP and its hydroxylated metabolities were reported to be much less toxic as compared to its parent compound diazinon [4]. Adenosine triphosphatases (ATPases) are a group of enzymes which play an important role in intracellular functions and critical for cellular        PHYSICAL CHEMISTRY 2014  F-05-P   462viability because they control many essential cellular functions, and are considered to be a sensitive indicator of toxicity [5, 6].  In the present study we investigated in vitro toxicity potential of various doses of diazinon and its degradation products (diazoxon and IMP) by determining the activity of AChE and ATPases (Na+/K+-ATPase and ecto-ATPase ) in rat brain synaptosomes which behave as minicells and represent a suitable model system for in vitro toxicity evaluation.  EXPERIMENTAL Synaptosomes were isolated from the brain of Wistar albino rats and incubated at 37oC for 1 hour in the presence of desired concentrations of diazinon, diazoxon and IMP. Activities of AChE and ATPases (Na+/K+-ATPase and ecto-ATPase) were measured by standard spectrophotometric methods [7, 8].  RESULTS AND DISCUSSION The influence of 1 hour exposure to increasing concentrations (within the range 10-9-10-4 mol/l) of diazinon and its degradation products (diazoxon and IMP) on the activity of synaptosomal AChE, expressed as a percentage of the control value (obtained without inhibitor), is presented in Figure 1. It is clearly apparent that inhibitor efficiency of diazinon, diazoxon and IMP is quite different. At the concentration of 1 × 10-5 mol/l diazoxon almost completely inhibited AChE activity (85%), while the same concentration of diazinon inhibited only 24% activity. The half-maximum inhibition (IC50, 1h) of the enzyme activity was achieved at (6.7 ± 0.2) × 10-8 mol/l of diazoxon, while the effects of the same concentration of diazinon as well as IMP on the enzyme activity were negligible. Moreover, the presence of the highest investigated diazinon concentration (1 ×10-4 mol/l) was not able to reach half-maximum inhibition. The dependence of Na+/K+-ATPase and ecto-ATPase activity on various concentrations (10-7-10-4 mol/l) of diazinon and its degradation products is presented in Figure 2 (a and b). The obtained results (Figure 2a) show that hydrolysis product of diazinon (IMP) did not remarkably alter Na+/K+-ATPase activity. Unlike IMP, diazinon and its oxidation products (diazoxon) inhibited the enzyme in concentration-dependent manner, but with various potencies. The presence of the maximal investigated diazoxon concentration (1 × 10-4 mol/l) induced almost complete inhibition, while the same concentration of its parent compound (diazinon) decreased the enzyme activity approximately 50% related to control (Figure 2a). The obtained results for ecto-ATPase (Figure 2b) show similar sensitivity of the enzyme toward diazoxon and IMP as in the case of        PHYSICAL CHEMISTRY 2014  F-05-P   463Na+/K+-ATPase, while diazinon did not cause ecto-ATPase activity inhibition more than 15 %.  10-9 10-8 10-7 10-6 10-5 10-402040608010023AChE activity (% of control)c (M)1 Figure 1. Inhibition of AChE activity in rat brain synaptosomes after 1 hour exposure to different concentrations of diazinon (1), diazoxon (2) and IMP (3).  CONCLUSION In the present work the activities of AChE, Na+/K+-ATPase and ecto-ATPase have been used to investigate neurotoxic effects of diazinon and its decomposition products. The results of our study show that diazinon and its oxidation product, diazoxon, inhibited synaptosomal AChE and ATPases activity in concentration-dependent manner but with varying potencies. Diazoxon demonstrated the strongest neurotoxic effect through the reduction of AChE activity, and the obtained inhibition of Na+/K+-ATPase and ecto-ATPase suggests ion exchange as well as purinergic signaling disrupting effect of the oxo analog of diazinon. On the contrary, IMP was found as a non toxic diazinon metabolite.  Considering the obtained changes of the physiologically important parameters, synaptosomes, which functionally behave as minicells, could be recommended as a suitable model system for in vitro toxicity evaluation of OPs and their degradation products.         PHYSICAL CHEMISTRY 2014  F-05-P   46410-7 10-6 10-5 10-4020406080100120(a)321Na+/K+ -ATPase activity (% of control)c (M)10-7 10-6 10-5 10-4020406080100(b)312ecto-ATPase activity (% of control)c (M)   Figure 2. The dependence of rat brain synaptosomal Na+/K+-ATPase (a) and ecto ATPase (b) activity on increasing concentrations of diazinon (1), diazoxon (2) and IMP (3).  ACKNOWLEDGEMENT  This work was financially supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Project No. 172023).  REFERENCES [1] F. Nachon, X. Brazzolotto, M. Trovaslet, P. Masson, Chem. Biol. Interact., 2013, 206, 536-544. [2] C. Cox, J. Pestic. Reform, 1992, 12, 30–35. [3] Q. Zhang, S.O. Pehkonen, J. Agric. Food Chem., 1999, 47, 1760–1766. [4] Y. Ku, J.L. Chang, S.C. Cheng, Water, Air, Soil Pollut., 1998, 108, 445–456. [5] E.O. Oruc¸ D. Usta, Environ.Toxicol. Phar., 2007, 23, 48–55. [6] M. Čolović, D. Krstić, S. Petrović, A. Leskovac, G. Joksić, J. Savić, M. Franko, P. Trebše, V. Vasić, Toxicol. Lett., 2010, 193, 9-18. [7] G.L. Ellman, K.D. Courtney, V. Andreas, R.M. Featherstone, Biochem. Pharmacol., 1961, 7,  88–90. [8] V. Vasić, D. Jovanović, D. Krstić, G. Nikezić, A. Horvat, L. Vujisić, N. Nedeljković, Toxicol. Let., 1999, 110, 95-104.  

In vitro evaluation of diazinon and its degradation products neurotoxicity potential in rat brain synaptosomes

http://vinar.vin.bg.ac.rs/bitstream/id/23094/471-474(1).pdf

In vitro evaluation of diazinon and its degradation products neurotoxicity potential in rat brain synaptosomes

Abstract

Similar works

Full text

Available Versions

Repository of the Vinča Nuclear Institute (VinaR)