J Arthropod-Borne Dis, June 2016, 10(2): 159–167 M Ghafourian et al.: The Effect of … 159 http://jad.tums.ac.ir Published Online: January 05, 2016 Original Article The Effect of Hemiscorpius lepturus (Scorpionida: Hemiscorpiidae) Venom on Leukocytes and the Leukocyte Subgroups in Peripheral Blood of Rat Mehri Ghafourian 1, Neda Ganjalikhanhakemi 2, Ali Asghar Hemmati 3, Rouhullah Dehghani 4, *Wesam Kooti 5 1Health Research Institute, Research Center of Thalassemia and Hemoglobinopathy, Department of Im- munology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 2Department of Pharmacology, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 3Department of Pharmacology and Toxicology, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 4Department of Environmental Health, School of Health and Social Determinants of Health (SDH), Re- search Center, Kashan University of Medical Sciences, Kashan, Iran 5Student Research Committee, Kurdistan University of Medical Sciences, Sanandaj, Iran (Received 2 Mar 2014; accepted 3 Dec 2014) Abstract Background: The aim of this study was to investigate the effect of Hemiscorpius lepturus venom on leukocytes and the leukocyte subgroups in peripheral blood of rat. Methods: In this experimental study, sixty N-Mari rats were divided into three groups of 20 rats. Then the rats in each group were divided into four subgroups based on the blood sampling time that was 2, 6, 24 and 48 hours after the venom injection, respectively. The control group did not receive anything, however, the first and the second ex- perimental groups received 0.1 and 0.01mg/kg of venom, subcutaneously. In accordance with a designated four sam- pling times, the blood sampling was carried out in three groups. After RBC lysis, the leukocytes and leukocyte sub- populations were determined and counted using appropriate hematological standard methods. Results: The leukocyte and the neutrophil count at two (P<0.05), six (P<0.01) and 24 (P<0.05) hours after the ven- om injection showed a significant decline compared with the control group, this decrease was significant at the dose of 0.1 mg/kg until 48 hours after the venom injection (P<0.05). The lymphocyte count showed a significant decline throughout the all hours of the experiment, compared with the control group (P<0.05). Conclusion: Leukocytes are probably affected by the cytotoxicity effect of the H. lepturus venom in a dose- dependent manner. This could be a wakeup call for the medical staff to perform quick and accurate treatment in the least time possible. Keywords: Hemiscorpius lepturus, Venom, Leucocyte, Rat Introduction Scorpion envenomation is one of the main problems in the public health system in many countries in the world. This involves 2.3 bil- lion inhabitants in the areas with the scorpi- on sting threat (Chippaux et al. 2008). In 2008, the annual incidence of scorpion stings was 1.200.000 leading to 3250 deaths (Chip- paux et al. 2008, Khoobdel et al. 2013). This number demonstrated a relative growth in a report released in 2012 and exceeded than annually 1,500,000 scorpion stings, however, the mortality rate due to the scorpi- on stings showed a significant decrease and fell into 2600 deaths per year (Jean-Philippe 2012). The highest number of scorpion sting in the world has been allocated to Iran after Mexico (Osnaya-Romero et al. 2001, Dehghani *Corresponding authors: Mr Wesam Kooti, E-mail: kweasm@yahoo.com J Arthropod-Borne Dis, June 2016, 10(2): 159–167 M Ghafourian et al.: The Effect of … 160 http://jad.tums.ac.ir Published Online: January 05, 2016 and Fathi 2012). The annually 42,500 scor- pion stings and 20 following deaths have been reported from 2001 to 2009, in Iran (Celis et al. 2007). In the Middle East, among the 52 known species of scorpions, the most dangerous scorpions are reported in Iran (Celis et al. 2007). The scorpion sting has been reported from all of the provinces of Iran, however, the most common inci- dence rates have been detected in Khuzestan, with incidences of 541 per 100000 individu- als (Dehghani et al. 2009, Rafizadeh et al. 2014). Hemiscorpius lepturus belongs to the Hemiscorpiidae family, and is the most med- ically important and a dangerous scorpion in Khuzestan, Iran and the world (Shahbazzadeh et al. 2007). Hemiscorpius lepturus has been responsible for 15 % of the scorpion sting bite cases, however, it is the leading cause of 89 % of deaths followed by the scorpion sting (Pipelzadeh et al. 2007). The lethality arising from this scorpion is approximately 60 times higher than the average for the re- maining venomous scorpion stings in the region (Pipelzadeh et al. 2007). The venom of H. lepturus leads to acute renal failure, thrombocytopenia and micro- angiopathic hemolytic anemia, known as the nephrotoxic, hepatotoxic and hemolytic com- plications of the scorpion venom (Valavi et al. 2008). Envenomation by H. lepturus is char- acterized by various local and systemic signs. The local signs vary from erythema to ne- crosis, while the patient feels no pain. On the other hand, the nephrotoxicity is the most im- portant systemic complication that if left un- treated could result in severe renal, cardiac and pulmonary failure (Pipelzadeh et al. 2007). Consequently, it may damage the intestinal lamina propria (Mojgan Heidarpour et al. 2011). The venom may induce severe patho- logical damages in target organs such as skin, blood cells, central nervous system (CNS), and cardiovascular system (Seyedian et al. 2010). It leads to an increase in liver enzymes Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT) and Alkaline Phos- phatase (ALP), indicating the severe hepatic damage (Pipelzadeh et al. 2006). Khodadadi et al. showed that the H. lepturus causes more RBC lysis and LDH increase, comparing with the complications of envenomation resulting from the stings of two other species of scor- pions, Mesobuthus eupeus and Androctonus crassicauda (Khodadadi et al. 2012). During a five- year study performed in Ahvaz, it was described that 90 % of patients whom were admitted in hospitals due to the general condition worsening, were stung by H. lepturus (Mir Dehghan et al. 2001). The immune system cells are distributed throughout the body, from the outer most points to the deepest organs and tissues, such as blood, bone marrow, thymus and spleen. Despite this diversity, the major group of immune cells is the peripheral blood leuko- cytes. These cells apply a comprehen-sive supervision on different organs and tissues through the regular circulation from blood to lymph, from inside of the vessels to the out- side and from the interior of the organs to the blood, resulting in protection of the body against the pathological factors (Vodjgani 2012). The results of an experimental study aiming the investigation of the effect of en- venomation by H. lepturus on the hemato- logical indices three days after injection of the venom suggested that the leukocyte number has been increased over the normal range, however, there was not any signifi- cant difference with the control group (Dehghani et al. 2012). An increase in the peripheral leukocyte count had been demon- strated during the investigation of the blood among H. lepturus scorpion sting victims (Chitnis et al. 1993). While comparative studies to investigate the effects of H. lepturus venom on hematologic parameters and vital organs of the body have been done, but so far, any experimental research on the effect of the scorpion venom on the immune sys- J Arthropod-Borne Dis, June 2016, 10(2): 159–167 M Ghafourian et al.: The Effect of … 161 http://jad.tums.ac.ir Published Online: January 05, 2016 tem cells and its various sub-groups in the early hours after the sting, and further-more determination and investigation of the period and the intensity of the envenomation have not carried out yet. Therefore, the aim of the present study was to investigate the effect of H. lepturus envenomation on blood leukocytes and their subgroups in the early hours after the sting. The results of this study can help physicians, health officials and the medical staff to fast and accurate treatment of the victims of H. lepturuss sting and prevention of compli- cations of the scorpion venom on important factors of the immune system. Materials and Methods Animals Sixty male rats from N-Mari species (weight range of 300–350 grams) purchased from Pasteur Institute of Iran (Tehran), were used during the study. The animals were kept in standard cages in animal house at the School of Pharmacy of Ahvaz Jundishapur University of Medical Sciences. Rats were housed in temperature-controlled rooms (22– 25 °C) with constant relative humidity (40– 70%) and 12h/12h light/ dark cycle before doing experimental protocols. The study was performed in accordance with the principles of laboratory care estab- lished by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. Venom Preparation The scorpion electroshocked venom was provided as a lyophylized powder from Pas- teur Institute of Iran, (Venom and Therapeutic Biomolecule Lab, Biotechnology Res. Center, Tehran, Iran). The concentration of crude venom protein was determined by using BradFord method (Bradford, 1976). The 0.1 and 0.01 concentrations of the venom pre- pared in distilled water. The injection vol ume was 0.1 ml. Grouping of Animals and Sampling The animals were randomly divided into three groups of 20 rats in each. The control group did not receive any thing; however, the first and the second groups received H. lepturus venom at the concentrations of 0.1 and 0.01 mg/kg/BW subcutaneously. There- after, the animals of each group (N= 20) were divided into four subgroups (N= 5), with respect of the four blood sampling time, that were two, six, 24 and 48 hours after the venom injection, the animals were then kept in separate cages. Experimental Studies The animals of each subgroup were anes- thetized with ketamine and xylazine (Alfasan, Holland). The blood samples were obtained from the animal’s heart amounted to 0.5–2 ml by syringe. Soon after the sampling, the blood was maintained in glasses containing the anticoagulant, EDTA (Ethylene-Diamine- Tetra-Acetic acid), and the leukocytes were counted using the diluent solution, Marcanu (RBC lysis buffer) and the Neobar slide (hemocytometer) by using the light micro- scope (Olympus, 3H-Z-Japan), In order to count and determine the leukocyte sub- groups (including neutrophils, lymphocytes, monocytes and eosinophils) the appropriate peripheral blood smears were prepared on the microscope slides and then fixed by means of the water-free methanol. Then Giemsa staining (Merck- Germany) was car- ried out by the diluted stain with the rate of 1/10. And finally, the differential counting was performed using a 100x lens microscope (Mahbod 2008, Mansouri et al. 2015). Statistical Analysis Data were analyzed using the SPSS ver. 13 (version 13, SPSS Inc, Chicago, IL) and the statistical tests of ANOVA and LSD. Data were considered significant statistically J Arthropod-Borne Dis, June 2016, 10(2): 159–167 M Ghafourian et al.: The Effect of … 162 http://jad.tums.ac.ir Published Online: January 05, 2016 when P< 0.05 as presented in Fig. 1–5. Results The Effect of H. lepturus Venom on Leu- kocyte Count The scorpion venom has led to a signifi- cant reduction of leukocytes in the venom- receiving group with the dose of 0.01 mg/kg at two and 24 hours after the injection, com- pared with the control group (P< 0.05). This decrease has been maximum of six hours after the injection (P< 0.01). After 24 hours from the injection, the effect of the venom has subsided; and after 48 h there was no any significant difference with the control group (P> 0.05). The toxicity effect of the venom is intensified with the increase of the injected venom concentration, so that in spite of a decrease in venom effect in the group receiving 0.01 mg/kg after 48 hours, the de- crease in toxicity effect is still significant in the group receiving 0.1 mg/kg compared with the control group (P< 0.01) (Fig. 1). The Effect of the H. lepturus Venom on the Peripheral Blood Neutrophil Count The neutrophil mean count showed a signifi- cant decline in the venom receiving group with the dose of 0.01 mg/kg at two, 24 and 48 hours (P< 0.05), and six hours after the injection, compared with the control group (Fig. 2). The Effect of H. lepturus Venom on the Peripheral Blood lymphocyte Count The mean of the peripheral blood lym- phocyte count in the venom receiving group of 0.01 mg/kg at two, six and 24 hours (P< 0.05), and 48 hours (P< 0.01) after the injec- tion demonstrated a significant decline com- pared with the control group, respectively. The Effect of H. lepturus Venom on the Peripheral Blood Eosinophil and Mono- cytes Count The comparison of the mean and standard deviation of the eosinophil and monocytes of the peripheral blood in the case and control groups showed no significant differences (P> 0.05) (Figs. 4, 5). Fig. 1. The comparison of the Mean±SD of white blood cells (× 10 per ml3) in peripheral blood of rat between the groups receiving venom with the con- centration of 0.01 and 0.1 mg/kg and the control group at different times * Significant difference (P< 0.05) between the experimental and control groups ** Significant difference (P< 0.01) between the experimental and control groups Fig. 2. The comparison of the Mean±SD of neutro- phils (× 10 per ml3) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg/kg and the control group at different times J Arthropod-Borne Dis, June 2016, 10(2): 159–167 M Ghafourian et al.: The Effect of … 163 http://jad.tums.ac.ir Published Online: January 05, 2016 * Significant difference (P< 0.05) between the experimental and control groups ** Significant difference (P< 0.01) between the experimental and control groups Fig. 3. The comparison of the Mean±SD of lympho- cytes (× 10 per ml3) in peripheral blood of rat be- tween the groups receiving venom with the concen- tration of 0.01 and 0.1 mg/kg, and the control group at different times * Significant difference (P< 0.05) between the experimental and control groups ** Significant difference (P< 0.01) between the experimental and control groups Fig. 4. The comparison of the Mean±SD of mono- cytes (× 10 per ml3) in peripheral blood of rat be- tween the groups receiving venom with the concen- tration of 0.01 and 0.1 mg/kg and the control group at different times Fig. 5. The comparison of the Mean±SD of eosino- phil (× 10 per ml3) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg/kg and the control group at different times Discussion The present study was aimed to inves- tigate the effect of H. lepturus venom on leukocytes and their subgroups in peripheral blood of rats. Leukocytes are considered as the major group of immune cells.These cells apply a comprehensive supervision on dif- ferent organs and tissues, through the regular circulation of blood to lymph, from inside of the vessels to the outside and from the inte- rior of the tissues to the blood, resulting in a protection of the body against the pathologi- cal factors. In general, leukocytes consist of various groups of cells including lympho- cytes, monocytes and granulocytes (Vodjga- ni 2012). The clinical syndrome induced by H. lep- turus sting is different from the stings by the other scorpions existing in Iran and the world, and exhibits more severe manifesta- tions. Lack of local pain or being a mild pain after the sting, cutaneous manifestations such as erythema, swelling and necrosis at the sting site, the red blood cell lysis, and ne- phrotoxicity, including hemoglobinuria, pro- J Arthropod-Borne Dis, June 2016, 10(2): 159–167 M Ghafourian et al.: The Effect of … 164 http://jad.tums.ac.ir Published Online: January 05, 2016 teinuria and hematuria are some manifestations among scorpion sting victims (Radmansh 1990, Radmanesh 1998, Pipelzadeh et al. 2006). The results of the present study demon- strated those leukocytes are affected by the venom after two hours, exhibiting sensitiv- ity, decrease in leukocytes continues for six hours after the venom injection. If leuko- cytes were evaluated after 12 hours, it was possible to notice a decreasing trend, due to prolongation of the presence of the venom into the body. Probably these variations in leukocytes are the results of cytotoxic effects of H. lepturus venom, which leads to white blood cell lysis and destruction (Shayesteh et al. 2012). There is no any considerable ex- perimental or clinical study in this field, and the decrease in leukocytes in the early hours is one of the new findings of this research. Lack of the related reports might be due to the difference in evaluation time of the he- matological indices in various studies (Dehghani et al 2005, Dehghani et al. 2012), it has been the result of the use of the anti- venom in human researches, as well (Chitnis et al. 1993, GhafourianBoroujerdnia and Mohebbi 2008). In the present study, the leukocyte count trend increased after 24 hours of venom injection, and there was not any significant difference in the venom-re- ceiving group of 0.01 mg/kg with the control group. The bone marrow has been probably ca- pable to reconstruct white blood cells. Vari- ous studies have reported leukocytosis as well as the major clinical signs of the enven- omation by H. lepturus, after other symptom such as hemoglobinuria, hematuria and pro- teinuria (Chitnis et al. 1993, Ghafourian Boroujerdnia and Mohebbi 2008). Therefore, the reconstruction of leukocytes observed in this study is in agreement with the previous findings (Dehghani et al. 2005, Dehghani et al. 2012). However, the more venom con- centration, the more decline in leukocyte count, the slower WBC reconstruction, so that the leukocyte count has not reached to the normal range after 48 hours of enven- omation with 0.1 mg/kg. According to the present study, after two hours, the percent of the blood neutrophils has been changed from the normal range of 61 % to 35.2 %. This reduction has been more severe after six hours and has fallen down to 25.5 %. Neutrophils make up the most abundant population of white blood cells, and mediate the primary stages of inflammatory response. They are the most effective phagocytes in peripheral blood and have a major role in defense against the extracellular factors (Abbas et al. 2011). These mature cells migrate to the inflammation site just four hours after the antigen entrance, and have the capability to invade the antigen. Phagocytosis of the particles and waste products in neutrophils is associated with a series of biochemical events and morphological changes in the cell (Vodjgani 2012). The majority of the neu- trophils at the inflammation site will be wiped out by other cells, such as macro- phages, after the phagocytosis of the invader. Therefore, this can explain the rapid dec- rease in neutrophils. Jalali et al. (2011) aiming to investigate the effect of H. lepturus on the serum levels of cytokines IL-1β, IL-6, IL-8 and TNF-α, demonstrated a direct relation between the worsening of the patient’s general condition and the above-mentioned cytokines. IL-8 is a chemotactic protein that is known as NAP-1 or the attractive and activator of neutrophils (Vodjgani 2012). The increase of this cyto- kine leads to fever and hyperthermia, which is probably due to neutrophil aggregation, pathogen killing and the death of the two kinds of cells (Taraz 2008). TNF-α enhances the production of some particular serum pro- teins such as amyloid A, through affecting hepatocytes. This cytokine suppresses stem cell division, which may lead to neutropenia (Vodjgani 2012). Therefore, the severe de- J Arthropod-Borne Dis, June 2016, 10(2): 159–167 M Ghafourian et al.: The Effect of … 165 http://jad.tums.ac.ir Published Online: January 05, 2016 crease in neutrophils in the group receiving high doses of H. lepturusvenom will be rea- sonable. In the following 24 hours, the neu- trophil count especially in the venom- receiving group of 0.01 mg/kg has app- roached the normal range. Regarding the neutrophilia, the neutrophil compensation could be explained. Through the neutrophilia the conversion of marginal neutrophils into circulating neutrophils could be noticed. These findings are consistent with the results of the research carried out by Ghafourian and Mohebby on patients with scorpion re- ferred to the hospital bite (Ghafourian and Mohebbi 2008). In the present study, lymphocytes have decreased during the first hours after the venom injection, and this reduction was sig- nificant even after 48 hours. One of the frac- tions extracted from the venom of H. lep- turus may reduce the lymphocyte count (Bigdeli et al. 2006). Lymphocytes make up around 20 to 40 % of the leukocytes, and 99 % of the cells in the lymph, respectively. Lymphocytes are considered as the most im- portant specific immune cells. The process of recognition and processing of antigen by lymphocytes as well as the clonal expansion requires the time, and the precise determina- tion of the mechanism of the effect of the venom of H. lepturus on lymphocytes cannot be explained during the first 48 hours, per- haps due to immunological reasons. In the present study, the blood lymphocyte de- crease was dose-dependent, which could probably be the result of the direct effect of the venom on the lymphocytes, which leads to disruption and lysis of these cells. In fact, it could be concluded that the venom of H. lepturus has a lymphotoxic effect (Ghafourian and Mohebbi 2008). In the present research, the peripheral blood eosinophil count in the control group showed no significant differ- ence with the experimental groups. Eosino- phil consists of 2 % of leukocytes found in the normal situation in tissues, especially the epithelium of the respiratory tract, gastroin- testinal tract and genitourinary tract. These cells have a weak phagocytic ability, and increase mainly in type-1 hypersensitivity responses and parasitic infections, as well. Therefore, the lack of their considerable change could be reasonable. Conclusion The venom of the scorpion H. lepturus ef- fect on the leukocytes in the early hours, as well as increasing the concentration of the toxin, its destructive power is increased. In other word, the venom effect is dose depen- dent. The scorpion venom may induce the release of bradykinin, prostaglandin and cor- ticosteroids. However, the essential role of these factors is to intensify the inflammation, but recently the natural corticosteroid hor- mones or similar synthetic substances are used to alleviate the inflammatory reactions against the allograft transplantation and im- mune system suppression. Therefore, the extraction of the useful fractions of H. lepturus venom for natural induction of corticosteroids and decreasing leukocytes may be useful in the treatment of some types of leukemia and the graft sur- geries, as well. It is recommended to investi- gate this issue in the future studies. How- ever, the severe and dose-dependent reduc- tion of the immune cells in the first hours after the injection of H. lepturus venom could be an alarm for health officials and medical staff to perform quick and accurate treatment in the least possible time, and to prevent of complications of the scorpion venom is the body's vital organs. Acknowledgements The results provided are from MD thesis of Neda Ganjalikhanhakemi approved with number\ 469 and implemented in the Student J Arthropod-Borne Dis, June 2016, 10(2): 159–167 M Ghafourian et al.: The Effect of … 166 http://jad.tums.ac.ir Published Online: January 05, 2016 Research Committee, and funded by the Re- search Deputy Vice-Chancellor for Research Affairs of AJUMS. The authors appreciate and thank this Deputy Vice-Chancellor for financial support, particularly the Research Consultation Center for technical support and Dr Delavar Shahbazzadeh and Miss Sara Ali-Akbari due to their unsparing coopera- tion. 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