CC BY
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UDC 616.151:591.111.1
V. P. Rosalovsky HEMATOLOGICAL INDICES OF RATS DURING THE FIRST HOUR AFTER CHLORPYRIFOS EXPOSURE
Institute of Animal Biology, National Academy Agrarian Sciences e-mail: [email protected]
We studied the effects of acute chlorpyrifos intoxication on the erythrocytes resistance to acid hemolysis and certain hematological parameters (number of erythrocytes, leucocytes, monocytes, lymphocytes, granulocytes, and trombocytes, hemoglobin content, hematocrit, trombocrit, and platelet indices).
Acute exposure to 50 mg/kg CPF leads to a decrease in the resistance of main erythrocytes pool to acid hemolysis at 15 minutes after exposure. The number of leucocytes increased by 21,9%, of erythrocytes - by 9,0%, hemoglobin content - by 6,3%, compared to controls, at 15 minutes after exposure. The number of trombocytes decreased by 21,6% at 15 min, by 26,2% at 30 min, by 53,3% at 45 min, and by 56,0% at 60 min after exposure, compared to control means.
Key words: hematological parameters, chlorpyrifos, rats, intoxication.
В.П. Росаловський ГЕМАТОЛОПЧН1 ПАРАМЕТРИ ЩУР1В ПРОТЯГОМ ПЕРШО1 ГОДИНИ П1СЛЯ ШТОКСИКАЦЛ ХЛОРП1РИФОСОМ
1нститут бюлоги тварин НААН e-mail: [email protected]
Фосфороргашчш сполуки (ФОС) у якосл основних дшчих агенпв входять до складу велико! ылькосп пестицидiв, 3ara6iB побутово! xiMi'i, використовуються у xiMi4Hrn промисловостi, медицинi (е компонентами препарапв що застосовуються для хiмiотерaпii туберкульозу i раку, мiaстенii, атони кишок, лiкувaння глаукоми). Висока токсичшсть та поширенiсть ФОС зумовлюе значну небезпеку iнтоксикaцiй цими речовинами. Хлортрифос (ХПФ) е одним з найпоширешших i нaйнебезпечнiших для здоров'я людини представником цього класу сполук. ХПФ (О,О-Диетил-О-3,5,6-трихлор-2-пiридилфосфоротiоaт (C9H11Q3NO3PS)), вiдомий головним чином, як дшча речовина багатьох розповсюджених iнсектицидiв широкого спектру ди. Основний мехaнiзм токсично! дп ХПФ полягае в тому, що вш шпбуе холiнестерaзнi ензими, спричиняючи, тим самим порушення синаптично! передaчi.
Дос^дження були проведенi на 40 статевозрьлих самцях бiлих лабораторних шурiв лшп Вiстaр масою тiлa 200-220 г, Було сформовано вiсiм груп тварин - чотири контрольних (К1, К2, К3, К4) i чотири дослЦш (Д1, Д2, Д3, Д4) по 5 шурiв у кожнiй. Декаштащю шурiв за анестезп ефiром та вiдбiр периферично! кровi у тварин проводили через 15 (К1, Д1), 30 (К2, Д2), 45 (К3, Д3), 60 (К4, Д4) хв. тсля введення в !х оргатзм хлорпiрифосу. Вiдiбрaну кров роздiляли на 2 частини. Для визначення гематолопчних пaрaметрiв зразки кровi вносили у пробiрки (Terumo Europe N.V. (Бельпя)), якi мiстили антикоагулянт ЕДТА-К2. Не шзшше нiж через 2 год. тсля взяття ISSN 2225-5486 (Print), ISSN 2226-9010 (Online). Бiологiчний вкникМДПУ. 2015. №1
124 Бюлопчний eicHHK Щ2 ---»
кров аналiзували на автоматичному гематолопчному аналiзаторi (Orphee Mythic 18, Швейцария) за наступними показниками: юльисть еритроцитiв, лейкоцитiв, лiмфоцитiв, моноципв, гранулоцитiв, тромбоцитiв, вмет гемоглобiну, гематокрит, тромбокрит, середшй тромбоцитарний об'ем i показник гетерогенносгi тромбоцитiв за об'емом. Дослiдження резистенгностi еритроцитiв до кислотного гемолiзу проводили на еритроцитах вЦмитих загальноприйнятим методом. Стшисть еритроцитiв до кислотного гемолiзу визначали за методом Терскова i Гiтельзона.
Встановлено, що гостра iнгоксикацiя щурiв ХПФ у дозi 50 мг/кг впродовж першо'1 години пiсля введения спричиняе: зростання илькосп лейкоцитiв на 29,1%, еритроципв - на 9%, вмiсту гемоглобiну - на 6,3% , порiвияно до контролю через 15 хв тсля штоксикацii.зменшения кiлькосгi тромбоцитiв через 15 хв- на 21,6%, через 30 хв-на 26,2%, через 45 хв - на 53,3% i через 60 хв - на 56,0% порiвняно з контрольними значеннями;зниження резистентносп основного пулу еритроципв до кислотного гемолiзу у груш Е1.
Ключовi слова: zeMamoxozi4m показники, хлортрифос, щурь ттоксикащя.
В. П. Росаловский.
ГЕМАТОЛОГИЧЕСКИЕ ПАРАМЕТРЫ КРЫС В ТЕЧЕНИЕ ПЕРВОГО ЧАСА ПОСЛЕ ИНТОКСИКАЦИИ ХЛОРПИРИФОСОМ Институт биологии животных НААН e-mail: [email protected]
Было проведено исследование влияния острой интоксикации хлорпирифосом на резистентность эритроцитов к кислотному гемолизу и некоторые гематологические показатели (количество эритроцитов, лейкоцитов, моноцитов, лимфоцитов, гранулоцитов, тромбоцитов, содержание гемоглобина, гематокрит, тромбокрит и тромбоцитарные индексы).
Острая интоксикация крыс ХПФ в дозе 50 мг/кг вызвала снижение резистентности основного пула эритроцитов к кислотному гемолизу через 15 минут после введения. Был зафиксирован рост количества лейкоцитов на 29,1%, эритроцитов - на 9,0%, содержания гемоглобина - на 6,3%, по сравнению с контролем, через 15 мин после интоксикации. Количество тромбоцитов снизилась на 21,6% через 15 мин, на 26,2% через 30 мин, на 53,3% через 45 мин и на 56,0% через 60 мин после введения, по сравнению с контрольными значениями.
Ключевые слова: гематологические показатели, хлорпирифос, крысы, интоксикация.
Introduction
Organophosphates (OPs) are the main active substances in many agriculture pesticides and household chemicals; they are also used in the chemical industry, medicine (tuberculosis and cancer chemotherapy preparations, drugs against myasthenia gravis, gastrointestinal atony, and glaucoma). Chlorpyrifos (CPF) is one of the most common and most dangerous of these compounds. CPF (O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate (C9H11Q3NO3PS)) is known as the active
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ingredient of many common broad-spectrum insecticides used in agriculture to protect vineyards, vegetables, citrus fruits and cereals from pests, and, that is particularly dangerous, to control insects in homes (Needham, 2005, Salyha, 2010a).
The main mechanism of CPF toxicity is inhibition of cholinesterase enzymes, causing disruption of synaptic transmission. Till recently, this was considered as a key and almost only toxicity mechanism of CPF, and all OPs in general, but the latest studies have convincingly shown that the toxic effect of OPs is more complex and is not limited to anticholinergic action. Besides cholinesterases, other potential molecular targets of OPs were found (Eaton, 2008; Salyha, 2010a; Salyha, 2010b; Abmali, 2011; Salyha, 2013). The free radical oxidation, a universal pathophysiological phenomenon in many pathological conditions, also plays a significant role in reaction to the toxic effects of various xenobiotics, including OPs and CPF in particular (Salyha, 2010b; Elsharkawy, 2013). Undoubtedly, in case of intoxication, blood is affected as well as other tissues, as haematological parameters are important markers of physiologic conditions of the body. Blood is a key homeostatic system of the organism (Savithri, 2010; Khaybullina, 2012; Salyha, 2013). Study of the blood system in context of homeostatic adaptive reactions to the toxic exposures is essential for understanding the pathologic mechanisms at cellular and tissue levels.
Under the effect of different compounds, particularly poisons, the nonspecific signs of alterations in peripheral blood mainly include changes in the total white blood cells number (mild leukocytosis) and leukocyte formula (lymphopenia, neutrophilia, sometimes with a left shift, eosinopenia or eosinophilia). In their turn, alterations in red blood are less prominent and are manifested mainly by a slight decrease in hemoglobin and erythrocytes number. Chronic exposure may lead to erythrocytosis with normal hemoglobin, leucopenia with left shift of the leucocytes formula, toxic granularity of neutrophils, and reduction of erythrocyte sedimentation rate (Khaybullina, 2012). Nevertheless, there is a relatively high variability of quantitative hemato-cytological parameters, depending on physicochemical features of the toxic factor, duration of its action, dose, intake route and so forth (Eaton, 2008; Savithri, 2010).
It is important that CPF poisoning (especially acute), besides other effects, can cause hypoxia (Salyha, 2010a). The performance stability of erythrocytes is often used in experimental medicine as a way to assess their functional state (Sybirna, 2010; Ambali, 2010). Unfortunately, the few available literature data on haematological parameters of animals under CPF action are often incomplete and even contradictory (Savithri, 2010). Therefore, the aim of this study was to investigate changes in basic hematological parameters and hemolysis resistance of the erythrocyte membrane at 15, 30, 45, and 60 min after CPF exposure to rats.
Materials and Methods
Forty adult male white Wistar rats weighing between 200 and 220g served as subjects for this study. Rats were housed under standardized laboratory conditions, with 12h dark/light cycle and free access to food and tab water ad libitum. All procedures were conducted according to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, 1986) and General Ethical Principles of Experiments using Animals (First National Congress of Bioethics, Kyiv, 2001).
The animals were randomly divided into eight groups: four control (C1, C2, C3, C4) and four experimental (E1, E2, E3, E4) groups, each comprising of five animals. All treatments were administered at a dose of 50 mg/kg. Administration of CPF was perfomed via oral gavage. Chlorpyrifos was diluted in sunflower oil. The control groups received the equivalent volume of pure oil.
At the end of the test period, the rats were sacrificed by decapitation after light ether anesthesia (at 15 min (groups C1 and E1), 30 min (C2 and E2), 45 min (C3 and E3), and 60 (C4 and E4) min after dosing, to obtain samples of peripheral blood.
The blood samples were collected into test tubes (Terumo Europe N.V., Belgium) with anticoagulant, K2-EDTA. Not later than 2 hours after sampling, blood was analyzed with the automatic hematological analyzer (Orphee Mythic 18, Switzerland) to examine for total red blood cells (RBC), white blood cells (WBC), lymphocytes, monocytes, granulocytes, platelets, haemoglobin (Hb) concentration, hematocrit, plateletcrit, mean platelet volume and platelet volume heterogeneity index.
Erythrocytes resistance to acid hemolysis was assessed by Terskov-Hitelson (GitePzon, 1959): photometric measurement of the decrease in the red blood cells number caused by adding 0.002N HCL in saline. The red blood cells were counted at regular intervals (30 sec).
The experimental data were processed by using the OriginPro 8 software. To compare between the data of the control and those of treatments, Student t-test was used. Values obtained were expressed as Mean±SD, Values of P<0.05 were considered significant.
Results and Discussion
Analyzing the hematological parameters of rat peripheral blood during the first hour after exposure to CPF, we found significant changes in the number of blood particles and in the acid hemolysis resistance of red blood cells. Below we present the CPF effects on parameters of blood particles number .
After the exposure, significant changes occurred in the total number of white blood cells. So, in the blood of group E1, i.e. at 15 min after CPF exposure, was observed a significant increase (by approximately 29,1%) in the number of white blood cells (p<0,05), compared with controls (Table 1).
On the other hand, at other studied time points, leukocyte count showed reversal dynamics: it tended to decrease by 10.37% in E2, and in the E3 and E4 groups it decreased significantly by 21,9% and 28,9% (p <0,05), respectively, compared to control values. Moreover, E1 group showed a significant increase in the number of granulocytes by 67,4% (p<0,05), compared with controls. No significant change in the number of monocytes, lymphocytes and granulocytes in groups E2, E3, E4 was observed. Increase of white blood cells in E1 may be related to their release from lymphoid-myeloid complex into blood to neutralize CPF-induced damage. The gradual decrease in E2, E3, and E4 may be related to the decrease in the oxidative stress-caused damage and completion of the disposal of damaged blood cellular components. These changes in characteristics of the white blood resonate with studies of other authors (Hissin, 1976), claiming that the number of white blood cells can change under the influence of organophosphorous pesticides. It is also reported that changes in the leukocyte number can be caused by CPF poisoning and depend on the severity of caused damage (Elsharkawy, 2013).
The number of platelets decreased significantly in all experimental groups, compared with intact animals. The intensity of these changes increased with time: the total platelet count decreased by 21.6% in group E1, by 26.2% in E2, by 53.3% in E3, and by 56.0% in E4, compared with control values. However, the rate of platelet volume distribution and average volume of platelets did not show any significant changes. The decrease in the number of thrombocytes can be caused by the formation of antigen-antibody complexes with platelet surface antigens, or by the changes in platelet membrane permeability, caused by oxidative stress (see, for instance, Sybirna, 2010; Ambali, 2011; Elsharkawy, 2013). It is known that changes in the platelet number can serve as an early sign of various intoxications.
Significant increase in number of red blood cells (by almost 9%, compared to the control group) was found only in group E1, i.e. 15 min after intoxication. We also observed here a significant increase in hemoglobin by 11% (p<0,05), hematocrit by 6,3% (p<0,05), compared to control. Such changes could be caused by hypoxia that occurs in the first few minutes after acute CPF poisoning, a powerful initiator of erythropoietin's synthesis and increased release of erythrocytes to the bloodstream from the depot, that initiate increasing hemoglobin content.. Within the same time intervals, erythrocyte count, total hemoglobin, the average volume of red blood cells showed no significant changes, compared to control.
Toxic substances affect the blood system and hematopoiesis in many ways: besides the bone marrow damage and abnormal hemoglobin transformation, they can also alter the degree of hemolysis of red blood cells. Violation of the erythrocyte membrane integrity, changes in surface properties of the lipid bilayer and protein conformation under the influence of toxic substances change the functional ability of erythrocytes to bind different compounds (Dudok, 2009; Ferents, 2014). Acid
erythrograms (Fig. 1) show the negative impact of CPF on the stability of erythrocyte membranes.
Fig. 1. Erythrograms of acid hemolysis and parameters of erythrograms of intact and exposed animals (1h after chlorpyrifos dosing)
In group E1, 15 min after CPF exposure, we observed decreased resistance to acid hemolytic of the main pool of red blood cells, showed by a significant increase in the ratio of hemolyzed red blood cells. In this group, total erythrocyte hemolysis occurred faster than in controls. At 30 min after intoxication (group E2), maximum red blood cells hemolysis percentage slightly decreased, compared with group E1, and a total destruction of red blood cells left unaltered. In Group E3 (45 minutes after experiment beginning) the percentage of damaged red blood cells significantly decreased up to 33.5%, compared to control (p<0,05). However, the increased area under the hemolysis curve of both right and left inflexions indicates the emergence of erythrocytes fraction with increased resistance to acid hemolytic (Fig. 1). After 60 min after CPF exposure, in group E4 the number of red blood cels destroyed by hemolytic approached the control values. In the peripheral blood of rats, the appearance of erythrocytes that are more resistant to acid hemolytic can be explained by substitution of the pool of destroyed erythrocytes with cells that are more resistant to hemolysis.
Acid erythrogram method helps to assess the condition of hydrophobic and protein components of erythrocyte membranes and to form morphologically homogeneous groups of erythrocytes by their age. The most resistant to hemolytic are the young red blood cells that are situated on the right side of the erythrogram. Aging of the erythrocytes is accompanied by a gradual decrease in their resistance, manifested in shifting the curve to the left.
Conclusions
An acute exposure to chlorpyrifos (50 mg/kg), 15 min after dosing) caused the increase in the white blood cells number by 29.1 percent, increase in red blood cells number by 9 percent, and increase in haemoglobin by 6.3 percent compared with control.
We also registered a decrease in the platelet number by 21.6 percent (15 min after dosing, group E1), by 26.2 percent (after 30 min, E2), by 53.3 percent (after 45 min, E3), and by 56.0 percent (after 60 minutes, E4), compared with control values. We registered decreased acid hemolysis resistance of the main pool of red blood cells in group E1 15 min after intoxication);
In conclusion, significant adverse changes in hematological parameters are reported to be associated with exposure to CPF, in this present study. This therefore suggest that exposure to CPF may be considered to be among the risk factors for the development of anaemic condition. Hence, exposure to this drug should be minimized.
References
Ambali, S.F., Abubakar, A.T., Shittu, M, Yaqub, L.S., Anafi, S.B., Abdullahi, A.
(2010). Chlorpyrifos-induced alteration of hematological parameters in Wistar rats: ameliorative effect of zinc. Res. J. of Envirom. Toxicol , 4(2), 55-66. Ambali, S.F., Ayo, J.O., Esievo, K.A. (2011). Hemotoxicity Induced by Chronic
Chlorpyrifos Exposure in Wistar Rats: Mitigating Effect of Vitamin C. Vet. Med. Int, 2011, 1-7.
Carlberg, I. (1975). Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J. Biol. Chem, 250, 5475-5480.
St
Dudok, K. P., Fedorovych, A. M., Dudok, T. G., Rechytskyi, O. N., Yeresko, V. A., Shkavolyak, A. V., Sybirna, N.O. (2009). Influence of spirocarbone and derivates of pyrrolopyrimidinedions on phisical and chemical properties of the hemoglobin ligand forms of blood in vitro. Studia Biologica, 3(2), 23-34.
Eaton, D. L., Daroff, R. B., Autrup, H., Bridges, J., Buffler, P., Costa, L. G., Coyle, J., McKhann, G., Mobley, W.C., Nadel, L., Neubert, D., Schulte-Hermann, R., Spencer, P. S. (2008). Review of the toxicology of Chlorpyrifos. Critical reviews in toxicology, 38(2), 1-125.
Elsharkawy, E.E., Yahia D., El-Nisr, N.A. (2013). Sub-chronic exposure to
Chlorpyrifos induces hematological, metabolic disorders and oxidative stress in rat: Attenuation by glutathione. Env. Toxic. and Pharm, (35), 218-227.
Ferents, I.V., Brodyak, I. V., Lyuta, M. Ya., Kulachkovs'kyi, O.R., Sybirna, N.O.
(2014). Effect of agmatine treatment on morphofunctional characteristics of the erythron in experimental diabetes mellitus in rats. Fiziol. Zh., 60(4.), 70-79.
Gitel'zon, I. I., Terskov, I. A. (1959). The erythrogram as a method of clinical investigation of the blood. Krasnoyarsk: Scientific academy of USSR, Siberia department.
Hissin, P.J., Hilf, R.A. (1976). A fluorometric method for determination of oxidized and reduced glutathione in tissues. Analyt. Biochem, 74(21), 214-226.
Khaybullina, Z.R, Vahidova, N.T. (2012). Parameters of peripheral blood under experimental acute hypoxia. Chelyabinsk: Dva Komsomoltsa, (35), 24-29.
Needham, L.L. (2005). Assessing exposure to organophosphorous pesticides by biomonitoring in epidemiologic studies of birth outcomes. Environ. Health Persp, 113(43), 494-498. Salyha, Y. (2010a). Biological effects assessment of chlorpyrifos and some aspects of
its neurotoxicity. Visnyk of Lviv University. Biology series, 54, 3-14. Salyha, Y. (2010b). Toxic effect of chlorpyrifos on hippocampal neurons in vitro. The
Animal biology, 12(1), 163-168. Salyha, Y. (2013). Chlorpyrifos leads to oxidative stress-induced death of
hippocampal cells in vitro. Neurophysiology, 45(3), 193-199. Savithri, Y., Sekhar, P.R., Doss, J.P. (2010). Changes in hematological profiles of
albino rats under chlorpyrifos toxicity. Inter. J. Pharma and Bio Sci., 1(3), 1-7. Sybirna, N.O., Lyuta, M.Ya., Klymyshyn, N.I. (2010). Molecular mechanisms of nitric oxide deposition in erythrocytes. Studia Biologica, 4(1), 143-160.
Поступила в редакцию 02.04.2015 Как цитировать:
Rosalovsky, V.P. (2015). Hematological indices of rats during the first hour after chlorpyrifos exposure. Биологический вестник Мелитопольского государственного педагогического университета имени Богдана Хмельницкого, 5 (1), 123-132. erossrcf http://dx.doi.org/10.7905/bbmspu.v5i1.968
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ISSN 2225-5486 (Print), ISSN 2226-9010 (Online). Бюлогiчний вкникМДПУ. 2015. №1
