BIOCHEMICAL AND HAEMATOLOGICAL CHANGES IN PERIPHERAL BLOOD OF RATS EXPOSED TO CHLORPYRIFOS..

Chlorpyrifos is a highly toxic organophosphate compound. It is still among the most widely used insecticide, and the main mechanism of its toxicity is associated with inhibition of cholinesterases. A long with the anticholinesterase action, CPF may affect other biochemical mechanisms, particularly through disrupting proand antioxidant balance and inducing free-radical oxidative stress. We studied the action of A and E vitamins on the basic haematological and biochemical parameters of rat peripheral blood after 12 hours of a single chlorpyrifos intoxication. Exposure to 70 mg/kg chlorpyrifos caused a decrease in the total number of red blood cells (RBCs), platelets, and total haemoglobin content. We also observed a decrease in the acid haemolysis resistance of RBCs in peripheral blood of CPF-poisoned rats. Combined exposure to chlorpyrifos and vitamins A and E caused changes in haemolysis resistance of RBCs, approaching the control values. In addition, it was found that chlorpyrifos intoxication disrupt prooxidant-antioxidant balance as evidenced by the increase of lipid peroxidation products: lipid hydroperoxides and thiobarbituric acid reactive substances. However, administration of vitamins A and E during intoxication provided levelling effect on the formation of lipid peroxidation products. CPF intoxication caused an increase of the catalase activity, while superoxide dismutase, glutathione peroxidase and glutathione reductase activities and the content of reduced glutathione decreased. It was revealed that combination of vitamins A and E cause corrective effect at the platelets quantity, and lipid hydroperoxides thiobarbituric acid reactive substances amount of rat peripheral blood.


INTRODUCTION
Organophosphorous compounds (OPs) belong to a group of phosphonic or phosphoric acid derivatives, widely used in agriculture, primarily as components of insecticides, defoliants and means against animal ectoparasites. The most frequent causes of acute OP poisonings are violation of personnel safety regulations, accidental intake, and suicidal attempts [13]. animals of groups E2 and E4 were administered of vit. A and E solution 5 minutes later after CPF exposure. All exposures were conducted intragastrically via oral gavage. The control group received a corresponding amount of pure oil. After 12 hours of CPF, animals were euthanized with ether anaesthesia and decapitated and peripheral blood was collected.
For study of haematological parameters, blood samples were put in test tubes (Teru mo Europe NV (Belgium)) containing as anticoagulant EDTA-K 2 . No later than two hours after sampling, blood was studied in automatic haematological analyzer (Orphee Mythic 18, Switzerland). The following parameters were studied: the number of RBCs, white blood cells, lymphocytes, monocytes, granulocytes, platelets, haemoglobin concentration, haematocrit, and mean platelet volume and platelet volume heterogeneity index. RBC resistance to acid haemolysis was determined by Terskov and Gitel'zon [7].
For biochemical studies, heparinized blood samples were centrifuged for 15 minutes at 1,500 g. After plasma separation, erythrocytes were washed three times with 0.15 M NaCl solution. Haemolysates were obtained by three times freezing-thawing of aqueous suspensions of RBCs and their subsequent centrifugation at 10,700 g for 15 min.
Superoxide dismutase (SOD) (EC 1.1.15.1.) activity was determined by Dubinina et al. [5], based on the reduction of nitroblue tetrazolium to nitroformazan by superoxide anion, that are formed in the reaction between phenazine methosulphate and NADPH. SOD activity was expressed in arbitrary units per 1 mg protein.
Catalase (CAT) (EC 1.11.1.6) activity in the haemolysates was determined by Koroliuk [15] with modifications. The method is based on the ability of hydrogen peroxide to form a stable colored complex with molybden salts. Enzyme activity was expressed in mmol H 2 O 2 /min per 1 mg protein using a molar absorption coefficient 22200 M -1 cm -1 .
Glutathione peroxidase (GPO) (EC 1.11.1.9) activity was determined by the rate of GSH oxidation before and after incubation with tertiary butyl hydroperoxide. This colour reaction is based on the interaction between SH-groups with the 5,5'-dithiobis(2-nitrobenzoic acid) (DTNBA), resulting in a colored product, thionitrophenyl anion [18]. Quantity of the thionitrophenyl anion is directly proportional to the number of SH-groups reacted with DTNBA. Enzyme activity was expressed in μmol GSH/min per 1 mg protein.
Glutathione reductase (GR) (EC 1.6.4.2) activity was determined by Carlberg [3]. This method is based on the catalytic NADPH-dependent reduction of oxidized glutathione form, the intensity of which can be measured by the rate of extinction decline on the wavelength of NADPH maximum absorption 340 nm. Calculation of GR activity was carried out using molar absorption coefficient for NADPH 6200 M -1 cm -1 . The activity of the enzyme was expressed in μmol NADPH/min per 1 mg of protein.
The concentration of reduced glutathione (GSH) was measured spectrophotometrically before and after the reaction, by Hissin [11]. The colour reaction is based on the interaction between SH-groups of GSH and DTNBA. The GSH content was calculated with the calibration curve.
The content of lipid hydroperoxides (LP) in erythrocyte mass was determined by [27], based on spectrophotometric measurement of optical density of products formed in the reaction between ammonium thiocyanate, Mohr's salt and hydrochloric acid.
The concentration of thiobarbituric acid reactive substances (TBARS), characterizing the LPO rate, was determined by Korobeinikova, based on the reaction between malondialdehyde (MDA) and thiobarbituric acid (TBA), in conditions of high temperature and acidic environment, with formation of a colored trimethyl complex consisting of one MDA and two TBA molecules [14].
Protein concentration was determined by Lowry method [16]. All reagents used were obtained from Sigma-Aldrich and Fluka (USA). Obtained data were analyzed statistically with Student's t-test, using the program OriginPro 8. Data considered statistically significant at p <0.05.

RESULTS AND DISCUSSION
The basic integral marker of OP intoxication is a decrease in cholinesterase activity. The phosphorylation of cholinesterase leads to loss of its ability to hydrolyze acetylcholine. Because of this, we studied cholinesterase activity in blood plasma of all groups of animals ( Fig. 1).
Notably, in the blood plasma of E3 animals, ChE activity decreased only by 14 %, comparing with the intact animals. No significant differences in ChE activity were found between E1 and E3 groups.
On the next research stage, we studied the haematological parameters of peripheral blood. The results are presented in Table 1.
At 12 th hour after CPF exposure, a significant decrease in platelet count was observed in the peripheral blood of E1 group rats, which are consistent with results of our previous studies [19]. In addition, in this group was found a significant decrease in the number of RBCs (by 14 %) and haemoglobin content (by 8 %), compared with control values. According to S. F. Ambali, this decrease may be caused by the formation of cross-links between proteins and lipids of the cell membrane and inactivation of enzymes located in the cell. By-products of LPO can cause violations in composition and structure of membranes, extrude essential fatty acids, and inactivate membrane-bound enzymes. [2].  We did not found any significant changes in the total amount of leukocytes, lymphocytes, granulocytes, monocytes in E1, E2, E3 groups, compared to control. In the E2 group, the platelet number slightly decreased, but this decrease was not statistically significant. In the E3 group was observed a significant increase in the total platelet number, compared to E1 group.It is possible, that increase total quantity of platelets may be associated with antioxidant properties of both vitamins. Being well known antioxidants, vitamin E and A prevent the free-radical mediated damage of the platelets [1,2].
We have not detected significant intergroup differences between E1 and E3 groups. Minor changes in the number of RBCs in peripheral blood may be associated with changes in physical and chemical properties of erythrocyte membranes and duration of their life under CPF intoxication.
Moreover, vitamin E influences the cellular response to oxidative stress through modulation of signal-transduction pathways. According to S. F. Ambali [2], these pathways may provide the cellular regulation level. Lipid peroxidation can cause changes in rheological properties of erythrocyte membranes and membrane potential, increase their permeability for different ions that can initiate the process of haemolysis, and thus reduce the lifespan of erythrocytes [1].
To assess the stability of erythrocyte membranes, we studied their resistance to acid haemolysis (Fig. 2). The results are presented in Tab. 2.
Studying the acid haemolysis resistance of erythrocytes, we found significant changes in the stability of erythrocyte membranes (Table 2).
We observed a significant increase in the percentage of maximum haemolysis in all experimental groups. Therefore, it increased significantly to 33.8 % in the E1 group, to 39% in the group E2, and to 33 % in E3, compared to control. We found no significant differences between E1 and E3 groups in the percentage of maximum haemolysis.  Table 2. It is known that CPF is able to generate ROS [2,22]. The newly formed ROS can increase, directly or indirectly, the passive permeability of membranes to potassium and sodium ions and cause violations in the erythrocyte osmotic balance, thus reducing the lifespan of these cells [6,25].
At the same time, the number of primary lipid peroxidation products significantly (p<0.05) decreased (32 %) in the erythrocyte haemolysates of the E2 group, compared with the ones of intact animals. In the group E3, exposed to the combination of vitamin mixture and CPF, we observed more slight decrease in the number of LP than in E2: by 15%, compared with control. At the E3 group amount of primary lipid peroxidation products significantly decreased by 39% compared with E1 group.
Changes in the content of TBARS were also found in the experimental groups (Fig. 3, II). So, the content of TBARS significantly increased in E1 (by 80 %) and E3 (by 49 %) groups, compared with control values. According to I. Amara and S. F. Ambali, the increase of lipid peroxidation products content shows the accumulation of hydrogen peroxide, nitrites, nitrates and other compounds, the amount of which exceeds the one that can be disposed in detoxification processes. These products can cause oxidative damage to membranes through interaction with polyunsaturated fatty acids (PUFAs) and haeme iron in the erythrocytes [1,2]. The catalase (CAT) and superoxide dismutase (SOD) activities are important indicators of the functional state of the erythrocyte antioxidant system. It is known that CAT is involved in utilization of hydrogen peroxide produced in the process of biological oxidation, decomposing it into water and molecular oxygen. In the presence of H 2 O 2 , it oxidizes low molecular alcohols and nitrites. SOD performs dismutation of superoxide radical into molecular oxygen or hydrogen peroxide [4].
We found a significant increase in CAT activity (by 34 %) in haemolysates of RBCs of E1 group, compared with the control (Fig. 4, I). Instead, SOD activity decreased significantly in erythrocytes haemolysate in groups E2 and E3, by 66 and 32 %, respectively (Fig. 4, II). The glutathione system which includes glutathione and glutathione-dependent enzymes, has one of the leading roles in the utilization of ROS and the redox potential stability. This is due to synergism of glutathione-dependent enzymes, their participation in regeneration of some low-molecular antioxidants and the ability of glutathione to neutralize active oxygen intermediates through direct interaction [22]. We studied the content of GSH, GPO, and GR after 12 hours of CPF intoxication (Fig. 5). The content of GSH decreased by 15 % in the E1 group, while in the group E3 it increased by 9 %, compared to the control. Data in this study also show that GSH content in the group E3 increased by 27 % if to compare with E1 group.
Activity of GPO in RBC haemolysates of E1 and E3 groups significantly decreased (by 70 % and 60 %, respectively), compared to the control values. GR activity decreased by 40 % in E1 and by 46 % in E2, compared with the control. Treatment of CPF-intoxicated rats with vit A and E significantly increased GR activity by 33 % in group E3 if to compare with E1 group.
The reduction-oxidation cycle of glutathione is involved in regulation of oxidative stress, when the stress level is low [1]. The decrease of GSH content in erythrocyte haemolysates of E1 group is obviously connected with its consumption to maintain redox balance, particularly, when CPF affects mitochondrial metabolism [26]. Instead, higher glutathione levels under conditions of the correction of the toxic CPF effects by the vitamin mixture may indicates a decrease in the intensity of LPO, and perhaps even normalization of oxidative processes in the structures of rat erythrocytes.

CONCLUSIONS
We found that acute exposure to 70 mg/kg CPF led to changes in haematological parameters: a decrease in the number of red blood cells, platelets, and total haemoglobin. After CPF exposure, a significant decrease in platelet count was observed in the peripheral blood of E1 group rats. In the E3 group we observed a significant increase in the total platelet number, compared to E1 group. We found slight corrective effect of vitamins A and E at the total number of platelets.
Besides, a decreased acidic haemolysis resistance in erythrocytes of CPF-intoxicated rats was found. The studied mixture of vitamins A and E showed a slight protective effect on these haematological parameters. The toxicant caused some biochemical changes, e.g. increase in the number of TBARS and lipid hydroperoxides content in the RBCs of rats after 12 hours of exposure. We found activation of LPO processes due to CPF intoxication and decrease in their intensity under the effect of vitamins A and E. We also found an increase in CAT activity, and decreased SOD, GPO, GR activities and GSH content.