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Myocardium showed massive myocytolysis with interstitial necrosis. > Bowels showed oedema of lamina propria. > Creatine kinase (CK) and lactate ...
Accepted Manuscript Histopathological changes induced by Hemiscorpius lepturus scorpion venom in mice Mojgan Heidarpour, Emna Ennaifer, Hamed Ahari, Najet Srairi-Abid, Lamia Borchani, Ghader Khalili, Hossein Amini, Amir Ali Anvar, Samir Boubaker, Mohamed El-Ayeb, Delavar Shahbazzadeh PII:

S0041-0101(11)00386-2

DOI:

10.1016/j.toxicon.2011.12.011

Reference:

TOXCON 4240

To appear in:

Toxicon

Received Date: 31 October 2011 Revised Date:

15 December 2011

Accepted Date: 16 December 2011

Please cite this article as: Heidarpour, M., Ennaifer, E., Ahari, H., Srairi-Abid, N., Borchani, L., Khalili, G., Amini, H., Anvar, A.A., Boubaker, S., El-Ayeb, M., Shahbazzadeh, D., Histopathological changes induced by Hemiscorpius lepturus scorpion venom in mice, Toxicon (2011), doi: 10.1016/ j.toxicon.2011.12.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

*Highlights

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Highlights

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> The histological studies in experimental mice injected with one LD50 of Hemiscorpius lepturus scorpion venom showed degenerative changes in the kidney with disorganized glomeruli and necrotic tubular. > Myocardium showed massive myocytolysis with interstitial necrosis. > Bowels showed oedema of lamina propria. > Creatine kinase (CK) and lactate dehydrogenase (LDH) enzymes significantly increased in the serum in 9 hrs. > Generation of the massive necrosis is due to the toxic effects of the hemolytic and phospholipase proteins existing in the venom.

*Manuscript Click here to view linked References

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Histopathological changes induced by Hemiscorpius lepturus scorpion venom in mice Mojgan Heidarpoura, Emna Ennaiferb, Hamed Aharic, Najet Srairi-Abidd, Lamia

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Borchanid, Ghader Khalilie, Hossein Aminif, Amir Ali Anvara, Samir Boubakerb, Mohamed El-Ayebd, Delavar Shahbazzadehf*

Institute of Standards and Industrial Researches of Iran, Karaj; bLaboratoire of Human and experimental

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a

Pathology, Pasteur Institute of Tunis, 13, Place Pasteur, Tunis, BP-74, 1002 Tunisia; cFood Hygiene sciences,

Pasteur, Tunis,

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Azad Islamic University, Tehran, Iran; dLaboratoire de Venin et Toxin, Institut Pasteur de Tunis-13, Place BP-74, 1002 Tunisia; ePasteur Institute of Iran, Immunology Department, Tehran-Iran;

f

Biotechnology Research Center, Pasteur Institute of Iran, Medical Biotechnology Group, Venom and Toxin

Lab., Tehran, P.O. Box:13169-43551, Iran.

author. E. mail: [email protected], Telefax: (+98) 21-66 48 07 80.

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*Corresponding

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ABSTRACT

Envenomation by Hemiscorpius lepturus (H. lepturus) is associated with local necrosis, followed by systemic manifestations. In this work the LD50 of H. lepturus venom were

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determined

by

subcutaneous

(SC)

injection

in

white

Balb/c

mice

(5mg/Kg).

Histopathological alterations in organs such as kidney, heart, liver, lungs, stomach and intestine were determined in 3, 6, 12 and 24 hrs following experimental (SC) envenoming injection of one LD50 of the venom in Balb/c mice. Histological studies showed degenerative changes in the kidney with disorganized glomeruli and necrotic tubular in 3 h and reached to its climax in 6 h. Myocardium showed massive myocytolysis with interstitial necrosis in 3 h and reached to its peak after 6 h past envenoming. Bowels showed oedema of lamina propria 1

ACCEPTED MANUSCRIPT and slight villous necrosis. The enzymatic activities of creatine kinase (CK) and lactate dehydrogenase (LDH) were significantly increased in the serum in 9 h. No necrotic lesion observed in lungs and liver. The results indicate that the venom of H. lepturus is a highly cytotoxic, and induces massive tissue damages in specific organs, starting from the heart and

Keywords:

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kidney as the first target in 3 hrs and ends to the bowels in 6 hrs post envenomation.

Hemiscorpius lepturus, scorpion venom, histopathology, creatine kinase (CK), lactate

dehydrogenase (LDH).

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1. INTRODUCTION

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There are at least twenty-three scorpion species in Iran from which twenty species belong to Buthidae family; two belong to Liochelidae and one to Scorpionidae (Prendini 1802, Vachon, 1966, Farzanpay, 1987, Fet, 2000). The latter, Hemiscorpius lepturus is spread from West to East in the Southern country, as well as South of Iraq (Pringle, 1960). This scorpion is

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endemic in Khuzestan, Southwestern Iran with hot and humid climate and is considered the most dangerous species in the region. Ten to 15% of total scorpion stings during the hot season and almost all cases of scorpion stings during winter are due to H. lepturus.

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Envenomation by H. lepturus is a major public health problem in Khuzestan, particularly in children (Mirdehghan and Motlagh 2001) and contributes to 95% of all mortalities in scorpion-stung individuals in the province (Radmanesh, 1998).

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Envenomation by H. lepturus is characterized by various symptoms such as pain, sweating, fever and hypertension and neurological disorders, and even with cutaneous reaction (Radmanesh, 1998). In the more severe cases, hematuria due to hematolytic effect of the venom may occur (Radmanesh, 1990).

Less information about the pharmacokinetics and pharmacodynamic characteristics of the venom of H. lepturus scorpion is available. In one study some toxic effects of H. lepturus 2

ACCEPTED MANUSCRIPT venom were studied in rat (Pipelzadeh, 2006). The results, however, suggest that the venom from H. lepturus is primarily a cytotoxic agent over cultured fibroblasts and possesses hemolytic, nephrotoxic and to some extent hepatotoxic activities (Pipelzadeh, 2006).

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The present study was undertaken in order to study the toxicity effects of H. lepturus venom in the experimental Balb/c mice. Histological damages to the heart, kidney, liver, intestine and stomach of the mice were assessed after subcutaneous (S.C.) injection of one lethal LD50

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of the venom. Alterations in LDH and CK enzymes in the mice serum were also determined.

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2. Materials and methods

2.1. Scorpion venom

Venom of Hemiscorpius lepturus scorpion from Khuzestan (Iran) were collected by the veterinarian service of the RAZI Vaccine Development and Serum Research Institute of Iran

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and kept frozen at -20°C in their crude forms until use.

2.2. Determination of toxicity in vivo

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To determine LD50, the Spearman-Karber’s method was employed (World Health Organization: WHO, 1981). Briefly, appropriate venom concentrations were prepared in PBS containing 1% BSA in order to obtain at least four points within the linear portion of the

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dose-response curve and to cover the full range between zero and 100% of induced animal mortalities with a symmetrical distribution in comparison with 50%. Toxicity in Balb/c white mice (average weight 18-20g) was tested by subcutaneous (SC) injection of 100 µl of the solutions, and the LD50 was determined by a lethality test 24 h after injection. Deaths were monitored in 24 h and LD50 was then calculated.

2.3. Histopathological studies in mice 3

ACCEPTED MANUSCRIPT Four groups of six Balb/c male mice were each subcutaneous (SC) injected with one LD50 of crude venom for pathological study. The fifth group was each intramuscularly (IM) injected with the same amount of venom concentration in order to study the morphological changes in the site of injection. The sixth group was interadermaly (ID) injected with one LD50 of crude

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venom to study the pathological alteration. One group of six mice which was assigned as control was injected with PBS by the same route of SC, ID or IM. Each mouse was injected with the volume of 100 µl of solution in all experimental groups. The mice were sacrificed by

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cervical dislocation in 3, 6, 12 and 18 hrs and the organs of lung, liver, heart, kidney, stomach and intestine along with the calf muscle from one group of mice that were injected in the

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muscle, were taken and fixed in 10% formalin solution. After 24-48 hours the sample of organs were dehydrated in a grade alcohol series and embedded in paraffin wax. Sections of 4-5µm thick were stained with haematoxylin-eosin (H&E) for pathological studies based as

2.4. Enzyme assays

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described by Kiermann (1999).

The myotoxic activity was measured by determining creatine kinase (CK) and lactate

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dehydrogenase (LDH) activities in the serum of the mice described in section 2.3. The serum was obtained from the centrifugation of the blood samples collected from the orbital sinus of

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mice after 2 h incubation at 37°C. The creatine kinase (CPK reagents, Bayer commercial Kit, code SFBC: LO, BIO-2407-83, USA) and lactate dehydrogenase (LDH reagents, Bayer commercial Kit, code SFBC: BO, BIO-2111-83, USA) were assayed in the sera using Bayer commercial Kit according to the manufacturer’s instructions. The enzyme values were expressed in international units (IU/l).

2.5. Protein estimation

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ACCEPTED MANUSCRIPT Protein concentration was determined according to the method of Bradford et al. (1976), using BSA as standard.

2.6. Statistical analysis

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The results are reported as the means ± SEM of n experiments, when appropriate. The significance of differences between means was evaluated by analysis of variance followed by Student’s t-test when various experimental groups were compared with the control group.

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The confidence limit for significance was 5%.

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3. Results 3.1. LD50 determination

The LD50 amounts of H. lepturus venom were determined 5mg/Kg by SC injection in white

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Balb/c mice.

3.2. Histopathology and cellular changes

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The histological analysis of some organs from mice after subcutaneous exposition of one LD50 dose of the H. lepturus venom for 0, 3, 6, 12 and 24 hrs revealed remarkable alterations in the kidney, heart, intestine and stomach tissues. After 24 h post histopathology studies, the

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renal showed degeneration in the Bowman’s space, glomerular and tubular epithelial cells, with no deposition of eosinophilic material inside the proximal and distal renal tubules (Fig. 1 athological effect of H. lepturus venom on heart muscle was studied as intravascular fibrin network deposition, thrombosis of the dermal blood vessels, and degeneration of the blood vessel wall. The muscular edema was also present with myonecrosis of myofibrils (Fig. 1

iver, but it seems that it is suffering from the

envenomation (Fig. 1

H. lepturus venom in 5

ACCEPTED MANUSCRIPT Balb/c mice in the site of injection (Fig. 1 changes including oedema of lamina propria and slight villous necrosis (Fig. 2 a). After shivering and stretch attend postures of the animal, physical discomfort rendered to diarrhea

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in final state which macroscopically was obvious (Fig. 2 b).

3.5. Enzyme assays

The enzyme assays showed a significant increment in the level of CPK and LDH activities in

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the serum of envenomed mice compared to the controls (Fig. 3). At 6 h post envenomation,

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all treated animals showed moderated enzyme activities reached to severe states in 9 h.

3.6. Route of injection

As shown in Table 1, different ways of H. lepturus venom injections in Balb/c mice were performed. With the route of venom administration by SC, significant effect on the toxicity of

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injected mice was observed. Histopathological studies showed sever injury of the heart and kidney organs in affected mice in 3 h post envenomation. Tissue damage was completed with degeneration of stomach and intestines tissues in 6 h. These findings were observed in the

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group of mice that injected by IM route after 24, and moderate degeneration happened in ID route injected mice after 48 h. The lungs showed no injury by different routes of injection.

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3. Discussion

Clinical data for human envenomation by Hemiscorpius lepturus (H. lepturus), one of the dangerous scorpions in the south of Iran-Khuzestan, is associated with local necrosis, initially painless,

followed by systemic manifestations including intravascular haemolysis,

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ACCEPTED MANUSCRIPT coagulopathy and acute renal failure/damage (Radmanesh, 1998). The mechanism by which this venom induces the clinical signs is unclear. In the present study, we investigated the toxic effects induced by the venom of H. lepturus in mouse model. To achieve this aim, histopathogical and biochemical methods in Balb/c were

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used to assess these toxic actions.

The LD50 of H. lepturus was obtained 5mg/Kg which is in accordance with the result obtained in other study with the amount of 5.6 mg/Kg and by administration of one LD50

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dose of the venom. Also, they studied the histopathological manifestations of H. lepturus venom on rats with focal necrosis, hemorrhages, with and without edema in lipid-containing

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organs such as liver, kidney and spleen of the rats (Latifi and Tabatabai, 1979). In our study the kidney tissues showed degenerative changes with disorganized glomeruli, and necrotic tubular post envenomation by H. lepturus. Acute renal failure also has been cited by several authors as one of the main complications and most frequent cause of death in

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humans and animals envenomed with H. lepturus scorpion. The envenomation by H. lepturs scorpion induced uremic syndrome in a seven year-old female child (Valavi et al., 2008). Renal failure was reported in 7 cases out of 4485 patients with clinical symptoms due to the

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sting of H. lepturus after local necrosis. Following local necrosis appearing in the patients stung by H. lepturus, the systemic dysfunction begins and results in renal failure

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(Shahbazzadeh et al., 2009). Renal involvement and nephrotoxicity has been demonstrated in patients following H. lepturus envenomation that are clinically important as demonstrated by the development of proteinuria and presence of intact RBCs in the urine. These findings correlate with the histopathological findings in rats following experimental envenoming with H. lepturus (Pipelzadeh et al., 2006; Correa et al., 1997). In these studies, inflammation becomes evident, which did not occur in the mouse from our experiment. Dehghani et al. (2004) in histophatological examination of H. lepturus venom sting on rat, morphological

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ACCEPTED MANUSCRIPT changes observed in proximal tubular cell and glomerular network of the kidney. The phenomenon of kidney failure also was observed in the venom of Loxosceles the ubiquitous spider in temperate and tropical regions of North, Central and South America, Europe and Africa. The injury occurs with some modifications of hyalinisation and erythrocytes in the

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Bowman’s space, glomerular collapse, tubular epithelial cell cytotoxicity and deposition of eosinophilic material within the tubular lumen in C57Bl/6 mice (Tambourgi et al., 1998). Ultrastructural studies showed glomerular epithelial and endothelial cell cytotoxicity,

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alterations of the basement membrane and tubular epithelial cell degeneration (Luciano et al., 2004).

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Heart is another organ that suffers from scorpion envenomation. In our study the heart muscles showed massive degradation post envenomation by H. lepturus comparing to the other organs. The myocardium showed massive myocytolysis with interstitial necrosis in 3 h and reached to its peak after 6 h past envenoming. The cardiac damage and cardiovascular

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dysfunction following severe scorpion envenomation can be due or enhanced by the depressive effect of the cytokines on the myocardial cells (Bahloul et al., 2005). At necropsy done on a died 4 years old boy, a few hours after he had been stung by Tityus serrulatus

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scorpion, there were multiple foci of coagulative myocytolysis in the myocardium and pulmonary oedema. Myocardial necrosis was probably associated with the sympathetic storm

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induced by scorpion envenomation, and may have contributed to cardiac failure and death (Benvenuti et al., 2002).

The cytotoxicity was also observed in stomach and intestine other than kidney and heart organs in mice after 24 h of envenomation by H. lepturus (Fig. 2 a). In some cases diarrhea was also observed macroscopically in few hours post envenomation. It means that the toxin is able to spread in sensitive organs such as intestines and rapidly damages the digestion system which is a unique characteristic of this venom. There are no reports of such alterations in

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ACCEPTED MANUSCRIPT other animals inoculated with any kind of venomous animals except for that of brown spiders (Loxosceles genus) (Babcock, et al. 1981; Smith, et al. 1968). The organs of lung and muscle were also subjected to microscopic analysis; however, no

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significant alteration was found in the first one (Fig. 2 c), but some myonecrosis was

the venom of H. lepturus can not induce lung failure in mouse model.

At least three-fold increase in CK and LDH enzymes in our study suggests that H. lepturus

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venom produced cytotoxicity. The cytotoxic activity of the venom was confirmed in the other studies. Significant increase of the levels of CK, LDH, SGPT and SGOT specific enzymes in

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the serums of more than 200 child victims of H. lepturus envenomed patients were observed. These patients were being referred to the pediatric emergency department of Abozar Children’s Hospital in Khuzestan province of Iran 24 h following H. lepturus sting. Changes in the level of these enzymes may be used as a prognostic tool in human victims (Jalali et al.,

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2010). Two fold increase in transaminase enzymes (AST, ALT) alongside with ALP indicates the hepatotoxicity in both parenchymal and billiary tracts (Pipelzadeh, et al., 2006). The significant alteration in the plasma concentrations of LDH, SGOT, SGPT and CK enzymes

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following H. lepturus stings have not been observed in the studies of other scorpions (Hering et al., 1993, Amaral et al., 1992; Sofer and Gueron, 1988). These results may support the

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notion that the different clinical picture observed following envenomation by H. lepturus scorpion i.e. a dramatic increase in the concentration of intracellular enzymes, may be due to direct tissue damage. However, it has also been suggested that the observed alterations in prognostic enzymes may be due to stimulation of chemical mediators which trigger their releases (Correa et al., 1997). Five possible routes for venom injection (intracerebroventricular: ICV; intravenous: IV; intraperitoneal: IP; intramuscular; IM and SC) could be used. The lowest LD 50 value is

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ACCEPTED MANUSCRIPT always obtained by the ICV route. The SC route gives the highest LD50 value (Schweitz H. 1984). Thus, this route should be used for antivenom potency estimation since it seems to be the most frequent way by which accidental scorpion envenoming occurs (Krifi et al., 1998). We also confirmed this finding that the distribution of H. lepturus venom in the mice

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depends on the route of injection, and severe degeneration happens by SC route. With injecting of one LD50 of H. lepturs venom in Balb/c by SC route, the organs of heart and kidney become degeneration in 3 h. After 6 h the other organs including stomach and

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intestines were severely degenerated. Similar delayed histopathological changes were found after 48 h post envenomation by ID route injection of H. lepturus venom. These finding could

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be attributed to toxicokinetic differences between SC, IM and ID ways of venom injection. While it is widely believed that the majority of the symptoms resulting from scorpion envenomation is due to the massive amounts of neurotransmitters that are release during the ‘‘autonomic storm’’ (Ismail, 1995; Freire-Maia et al., 1994), there is however, some evidence

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that the venom from H. lepturus has direct hemolytic, nephrotoxic and hepatotoxic actions (Pipelzadeh et al., 2007, 2006). Previously two peptides were isolated from the venom of H. lepturs: Hemicalcin a 33-mer basic peptide reticulated by three disulfide bridges which is

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active on ryanodine-sensitive calcium channels (Shahbazzadeh, 2007), and Hemitoxin an other 35-mer peptide displaces α-dendrotoxin from its site on rat brain synaptosomes with an

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IC50 value of 16 nM (Srairi-Abid et al., 2008). These two peptides represent only 0.2% of the venom proteins and seem with no import effects in envenomation. There were no inflammatory responses in the present study due to the following reasons: this effect was not observed, the duration of exposure of the venom with the immunity system was not enough time to initiate the immunity response to the venom in Balb/c mice. In conclusion, the results indicate that the venom of H. lepturus is a highly cytotoxic, and induces massive tissue damages in the organs, starting from the heart and kidney as the first

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ACCEPTED MANUSCRIPT target in 3 h and ends to the bowels in 6 h post envenomation. The generation of the massive necrosis by H. lepturus scorpion might be due to the toxic effects of some proteins with gelatinolytic, caseinolytic, and hyal-uronidase activities which already detected in the venom of H. lepturs (Seyedian et al., 2010). More studies are needed to characterize the toxic effects

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of the purified proteins from the venom of H. lepturus scorpion. There are many common properties of H. lepturs venom from that of Loxoscele spider venom, which can be a suitable

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model to characterize the biological activities of the purified proteins in H. lepturus venom.

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Acknowledgment

This research was supported by an ACIP (Action Commune Inter Pasteurienne) project granted by the Institute Pasteur of Paris in toxin and venom researches. We are indebted to Professor Hechmi Louzir, head of the Pasteur Institute of Tunisia, to Professor Mostafa

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Ghanei and Dr Mohamad Reza Siavashi, the head and research deputy of Pasteur Institute of Iran for their helpful advices. We also thank Dr Zakaria Ben Lasfar and his collaborators (Veterinary Laboratory, Pasteur Institute of Tunis) for providing laboratory animals and his

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helpful assistances.

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Vachon M., 1966. [List of scorpions known in Egypt, Arabia, Israel, Libya, Syria, Jordan, Turkey, Iraq, Iran]. Toxicon 4, 209-18.

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Figure

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b

× 200

24 H

× 200

CONTROL CONTROL

24 H

× 200

CONTROL

× 200

12 24 H

d

× 200 2200 00

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CONTROL

c

CONTROL CONTROL

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a

X××400 200 400

24 H

× 100

× 100

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Fig. 1. Sections of kidney, liver, heart, and muscle from mouse inoculated with H. lepturus venom. Light microscopic analysis of renal (a), heart (b), liver (c) and (d) skeletal muscle of

focal collapse of glomerular basement membrane, diffused vacuolar degeneration of proximal

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and distal tubular epithelial cells, neither leukocyte accumulation nor marginalization or

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deleterious degeneration and almost no s

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necrotic foci far from injected site.

Figure

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a

c

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b

Fig. 2. The pathological effects of H. lepturus venom on the bowels of mouse after 24 h of

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envenomation. (a) The bowels showed the most degeneration of the organs that observed macroscopically. (b) Intestine showed the most deleterious effects developing oedema of lamina

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effects observed in lung tissues.

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propria and slight villous necrosis shown microscopically. (c) No significant histopathological

Figure

1200 Control

900

LDH

600 300

4000 3000

3 9 Time (hr)

CK

1000

0 0

Control

2000

0 0

24

3

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CK activity (IU/I)

LDH activity (IU/I)

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9

24

Time (hr)

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poisoned mouse (Average ± standard deviation, n=6).

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Fig. 3. Effect of one dose of LD50 of H. lepturus venom on serum metabolic enzymes of

Table

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Table 1. The effect of time and the route of injection of H. lepturus venom on appearance of histopatholgical changes in Balb/c organs. The number of mouse used in each group is shown

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under fraction, and the number of death mice with histopathologic lesions depicted by (+);

Group Route of Elapsed

heart

Kidney

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subcutaneous (SC); intramuscular (IM); itradermal (ID).

bowels

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3

+5/6

2

SC

6

+6/6

3

SC

12

+6/6

4

SC

24

+6/6

5

IM

24

+5/5

6

ID

48

+3/4

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lung

+5/6

+0/6

+1/6

+0/6

+6/6

+6/6

+2/6

+0/6

+6/6

+6/6

+3/6

+0/6

+6/6

+6/6

+4/6

+0/6

+1/5

+5/5

+2/5

+0/5

+1/4

+0/4

+1/4

+0/4

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1

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injection time (h)

liver