J -Borne Dis, K Pourkhalili et al.: Cardiotoxic and …

Original Article Cardiotoxic and Arrhythmogenic Effects of Hemiscorpius lepturus Venom in Rats Khalil Pourkhalili 1, Euikyung Kim 2, Navid Reza Mashayekhy 3, Mostafa Kamyab 4, Seyed Mehdi Hoseiny 5, Reihane Evazy 6, Abbas Zare Mirakabadi 7, *Ramin Seyedian 8 1Department of Physiology, School of Medicine, Bushehr University of Medical Sciences, Bushehr, Iran 2College of Veterinary Medicine, Gyeongsang National University, Jinju, South Korea 3Department of Cardiology, Amir Kabir Hospital, Arak University of Medical Sciences, Arak, Iran 4Department of Biotechnology, Persian Gulf Research Institute, Persian Gulf University, Bushehr, Iran 5Department of Hematology and Oncology, Bushehr University of Medical Sciences, Bushehr, Iran 6Department of Biology, Jahrom Branch, Islamic Azad University, Jahrom, Iran 7Department of Venomous and Antisera Produstion, Razi Vaccine and Serum Research Institute, Hesarak, Iran 8Department of Pharmacology and Toxicology, Bushehr University of Medical Sciences, Bushehr, Iran

(Received 1 Feb 2014; accepted 22 June 2014) Abstract Background: Envenomation by Hemiscorpius lepturus is not painful and the clinical manifestations include bloody urine due to hemoglobinuria or hematuria, dermonecrotic reactions,cardiac arrhythmia and in minority of cases acute renal failure which may lead to death following disseminated intravascular coagulation in infants. Cardiac effects of envenomation by H. lepturus venom including inotropic, chronotropic and arrhythmogenic properties are not studied as now in rat hearts with Langendorff apparatus. Methods: Rat hearts were allowed to equilibrate in its buffer and cardiotropic plus arrhythmogenic effects induced by injection of different doses of H. lepturus venom were detected and recorded by computer acquisition based data in Langendorff apparatus. The neutralizing effects of Razi Institute and autonomic drugs were assayed in parallel studies. Results: Hemiscorpius lepturus venom (25 µg/100 l) treatment caused a negative inotropic (65.4 ± 3.2 versus 110.2 ± 3.4) and chronotropic effects (186.3 ± 4.2 versus 302 ± 6.3) in comparison to normal saline. Arrhythmogenic aspects including bradycardia, QRS widening and ST depression were induced by venom injection. Pre venom treatment (20 min) of Razi Institute antivenom (10 µl) neutralized cardiotropic effects but post venom injection (15 min later) had no therapeutic role. Pre (10 min before) and post (15 min after) injection of adrenaline (10 µl) neneutralized cardiotropic effects while pre venom injection (20 min) of propanolol (10 µl) had aggravating effects. Conclusion: Our study paves the way for further in vivo investigation of cardiovascular effects of this venom for finding suitable treatments instead of its ordinary antivenom medication in cardiogenic shock induced by the venom.

Keywords: Hemiscorpius lepturus, Contractility, Arrhythmogenicity, Envenomation

Introduction Scorpion envenomation is common in reactions and in minority of cases acute renal tropical and subtropical areas of the world failure which may lead to death following dis- including southwestern part of Iran espe- seminated intravascular coagulation in infants cially in Khuzestan province (Prendini 2000, (Radmanesh 1990, Jalali et al. 2010). This crea- Shahbazzadeh et al. 2009). Envenomation by ture is one of the most dangerous , H. lepturus belonging to family Hemiscorpiidae and responsible for 95 % of the scorpion- is not painful and the clinical manifestations associated mortalities in Iran, that is proba- in stung patients include bloody urine due to bly due to the various pathological enzymes hemoglobinuria or hematuria, dermonecrotic in its venom like Hemitoxin, Hemicalcin and

*Corresponding author: Dr Ramin Seyedian, E- mail: [email protected] J Arthropod-Borne Dis, K Pourkhalili et al.: Cardiotoxic and …

Heminecrolysin (Shahbazzadeh et al. 2007, Animals Srairi-Abid et al. 2008). Female Wistar rats (body weight 180–230 Generally there is only a few case reports g) were obtained from Jundishapur Medical on direct cardiotoxic effects of scorpion en- University breeding center (Ahvaz, Iran) and venomation in human beings (Gueron et al. placed in groups of three in PVC cages with 1967, Bawaskar and Bawaskar 1998, Shapira free access to water and hard food pellets in et al. 1998, Cupo and Hering 2002). How- house of Bushehr University of Med- ever previous studies on the envenomation ical Sciences. They were kept at 20 ± 2 °C by H. lepturus suggest that there are in vivo and maintained at 12 hours light-dark cycle arrhythmogenic and negative inotropic ef- starting at 7 AM. The experimental protocol fects in rabbits in spite of its small amount of used in this study conforms to the guidelines accumulation of radiolabelled venom follow- of the National Institute of Health (NIH). ing subcutaneous injections in animal mod- els during biodistribution study (Mirakabadi Langendorff perfused heart preparation et al. 2011, Seyedian et al. 2012). Rats were heparinized (300–400 IU) and In this study, the direct effects of H. lepturus anesthetized with sodium pentobarbital (50 venom on rat hearts were investigated with mg/kg intraperitoneally). After a bilateral thor- Langendorff apparatus. First, inotropic and acotomy, hearts were rapidly excised and chronotrpic changes upon time were evaluated after insertion of an aortic cannula perfused and then, neutralization properties of Iranian retrogradely at a constant perfusion pressure Razi Institute Antivenom and other drugs in- of 90 cm H2O with gassed (5% CO2, 95% cluding epinephrine, propranolol and atropine O2) Krebs-Henseleit buffer (pH 7.4) contain- were employed to analyze direct or indirect ing NaCl (118.5 mM), NaHCO3 (25 mM), cardiotoxic effects of this venom thoroughly KCl (4.7 mM), KH2PO4 (1.2 mM), CaCl2. promoting further in vivo studies. 2H2O (1.8 mM), MgSO47H2O (1.2 mM), D- glucose 11 mM) at 37 °C. An epicardial elec- Materials and Methods trocardiogram (ECG) was continuously rec- orded using two fine stainless steel elec- Drugs and chemicals trodes, one attached to the apex of the heart The lyophilized venom was acquired by and the other placed on the right atrium and direct electrical stimulation of H. lepturus a metal clip attaching to the aortic cannula as telsons (15 mV) in Iran. The venom was the reference electrode. Left ventricular pres- centrifuged, lyophilized and stored at -20 °C sure was recorded throughout the experiment until reconstitution by addition of Normal by a water filled latex balloon placed in the Saline in our laboratory. The Iranian pepsin left ventricle and connected to a pressure digested polyvalent antivenom from horse transducer (MLT 844) through a fluid filled plasma against 6 common endemic scorpions catheter. Volume of the balloon was adjusted (Odontobuthus doriae, Mesobuthus eupeus, to obtain end-diastolic pressure of 8–10 mm Androctonus crassicauda, Buthotus saulcyi, Hg at the end of stabilization period and un- Buthotus sach and Hemiscorpius lepturus) changed for the remainder of the experiment. (Latifi and Tabatabai 1979, Dehghani and The electrical and hemodynamic functions Fathi 2012) were purchased from Razi Vaccine of the heart were continuously monitored and Serum Research Institute. The neutralizing with a computer-based data acquisition sys- ability of the used batches was 26 LD50/ml. tem (PowerLab system with Chart 5 software, Adrenaline, atropine and propranolol were AD Instruments, Australia). Left ventricular purchased from Sigma (St Loius, MO). developed pressure (LVDP), rate pressure J Arthropod-Borne Dis, K Pourkhalili et al.: Cardiotoxic and … product (RPP), Max and Min dp/dt and heart Results rate were calculated. Coronary flow (CF) was measured by timed collections of the coro- Inotropic and chronotropic effects of nary effluent. Hemiscorpius lepturus venom Cardiotropic effects of various doses (25, Experimental protocol 50 and 100 µg ) of H. lepturus venom re- Hearts were allowed to equilibrate for 20 constituted in Normal Saline (100 µl) are min with its buffer. After the stabilization shown in Fig. 1–3 (n=3). period, different doses of H. lepturus venom Representative image of negative chrono- (25, 50 and 100 µg) dissolved in normal sa- tropic effects of different doses from H. line (100 µl) were infused directly acquiring lepturus venom with the same volume (100 cardiotropic, inotropic and arrhythmogenic µl) on isolated rat heart in Langendorrf ap- changes with Langendorff apparatus. Razi paratus upon time versus normal saline in- Institute multivalent antivenom (10 µl) dis- jection was depicted in Fig 1. Results present solved in Normal Saline (100 µl) was inject- mean ± SEM of 3 independent experiments. ed pre (20 minutes) and post venom (15 min) Reducing effects on the contractile po- treatment. In another set of experiments in- tency of the heart was induced shortly (two cubated venom (25 µg) with Razi Institute min later) following H. lepturus injection in antivenom (10 µl) was titrated to 100 µl with langendorrf apparatus.Venom caused a sta- normal saline for 30 min at room tempera- tistically significant decrease in heart contrac- ture to investigate its neutralizing effect tility (negative inotropic) and rate (negative against cardiac aspects of H. lepturus ven- chronotropism) for 40–60 min before ending om. In order to evaluate the neutralizing ef- the examination at 60 minutes. Coronary fects of β adrenoceptors and cholinoceptors flow was significantly decreased (7.83 ± drugs, propranolol (10 µl) in addition to 0.21 ml to 2.81 ± 0.62 ml) post venom injec- adrenaline (10 µl) and finally atropine (100 tion (25 µg) in our experiments. µl) were used pre and post venom injection. Coronary blood flow, left ventricular devel- The neutralizing capacity of Razi Institiute oped pressure, dp⁄ dtmax and heart rate were antivenom against cardiogenic effects of recorded and outflow samples were collected Hemiscorpius lepturus every 1 min initially and every 10 min se- Razi Institute antivenom (10 µl) was used quentially until the end of our experiments as pre (20 min) and post (15 min) venom (25 (60 min) to evaluate the coronary flow chang- µg) injection and its neutralizing effect against es induced by the venom. the venom were depicted in Fig. 4 A, B. Razi Institute antivenom pretreatment (20 Statistical analysis min before venom injection) or incubation with Results are expressed as mean ± SEM of venom for 30 min at room temperature sig- three experiments. ANOVA plus post hoc nificantly neutralized negative inotropic ef- test was carried out in cardiotropic exper- fects while post venom injection had no effect. iments. Values of P< 0.05 were considered In another study incubated venom and significant. antivenom (25 µg in 10 µl) for 30 min at room temperature was titrated with normal saline (100 µl) and injected to evaluate the neu- tralizing effects of this remedy on rat hearts (Fig. 4C). There was no bradycardia and neg- ative inotropic effects following H. lepturus J Arthropod-Borne Dis, K Pourkhalili et al.: Cardiotoxic and …

venom (25 µg) injection pre incubated with µl) following using of H. lepturus venom are the antivenom and the post treatment of represented in Fig. 6A, B. antivenom failed to counteract the cardiotoxic Adrenaline (10 µl) treatment 10 min be- effects of venom injection. fore and 15 min after venom injection neu- tralized negative inotropic effects upon time. Arrhythmogenic effects of Hemiscorpius According to these diagrams negative lepturus venom inotropic effects were significantly neutral- Venom perfusion prominently induced ized by this drug since there was no decreas- bradycardia, with widened QRS complexes ing of LVDP (left ventricular diastolic pres- and ST depression after passing 50 min in all sure) following venom injection upon time. the envenomed hearts as shown in Fig. 5A. In another experiment, pretreatment of pro- Normal heart rate and ECG parameter were pranolol (10 µl) resulted in additive negative induced by normal saline (100 µl) infusion to effects on LVDP in isolated rat hearts (Fig. serve as control (not shown). Antivenom ad- 7) and in our final examinations incubation ministration (10 µl), 20 min before venom of atropine (100 µl) as an anticholinergic drug injection had neutralized QRS widening and with our venom (25 µg) prior to injection neu- bradycardia induced by H. lepturus venom tralized its inotropic effects showing the (Fig. 5B). probability of its indirect mechanism (Fig. 8). Injection of adrenaline and atropine in our Pretreatment of isolated rat hearts with study counteracted the late arrhythmogenic propranolol, 20 min before venom injection effects induced by H. lepturus venom (data had no neutralizing effect on negative not shown). inotropic aspects as represented in this figure. Protection and aggrevating experiments Incubation of venom and atropine for 30 using adrenaline, atropine and propranolol min had no negative inotropic effects in Pre and post injection of adrenaline (10 isolated rat hearts upon time.

Heart rate changes induced by different doses of H. lepturus venom Beats (beats/minute)

Time (minutes)

Fig. 1. Heart rate changes by different doses of Hemiscorpius lepturus venom in rats in Langendorff apparatus

# Significantly different from normal saline at P< 0.05 J Arthropod-Borne Dis, K Pourkhalili et al.: Cardiotoxic and …

Inotropic changes induced by doses of Hemiscorpius lepturus on isolated rat hearts (n= 3) ) Left Left Ventricular Developed Pressure ( LVDP (mmHg)

Time (min)

Fig. 2. Contractility changes induced by Hemiscorpius lepturus venom in isolated rat hearts

# Significantly different from normal saline at P< 0.05 ##Significantly different from normal saline at P< 0.01 mmHg) LVDP LVDP (

4 min Venom (25 µg)

Fig. 3. Inotropic effects induced by injection of Hemiscorpius lepturus venom (25µg) in rat hearts with Langendorff apparatus

A B mmHg) mmHg) LVDP LVDP ( LVDP LVDP (

Antivenom (10 µg) Venom (25 µg) Venom (25 µg) Antivenom (10 µg) 4 min 4 min J Arthropod-Borne Dis, K Pourkhalili et al.: Cardiotoxic and …

C Cardiac effects of scorpion venom (25 µg) incubated with antivenom (10 µl) for 30 minutes

Time (minutes)

Fig. 4. Inotropic changes following pre and post venom (25 µg) injection of Razi Institute antivenom (10 µl) A voltage (mV) voltage 15 minutes before venom injection 1 Sec

voltage (mV) voltage 10 minutes following venom injection 1 Sec

voltage (mV) voltage 30 minutes following venom injection 1 Sec voltage (mV) voltage 50 minutes following venom injection 1 Sec B

voltage (mV) voltage 15 minutes before venom injection 1 Sec voltage (mV) voltage 10 minutes following venom injection 1 Sec

voltage (mV) voltage 30 minutes following venom injection 1 Sec

voltage (mV) voltage 50 minutes following venom injection 1 Sec Fig. 5. Arrhythmogenic affects of Hemiscorpius lepturus venom injection (25 µg) upon time (5A) versus normal saline (5B). Bradycardia, QRS widening and ST depression were induced with venom injection in Langendorff apparatus J Arthropod-Borne Dis, K Pourkhalili et al.: Cardiotoxic and …

A B mmHg) mmHg) LVDP LVDP ( LVDP LVDP (

4 min Epi (10 µl) Venom (25 µg) Venom (25 µg) Epinephrin (10 µl) 4 min Fig. 6. Changes in inotropic effects upon the pre and post treatment of epineprine (10 µl) added before and after venom (25 µg) injection mmHg) LVDP LVDP (

Propranolol (10 µl) Venom (25 μg) 4 min

Fig. 7. LVDP changes following pretreatment with propranolol (10 µl), added 20 min before venom (25 µg) injection mmHg) LVDP LVDP (

Atropine (100 µl)+ Venom (25 µg) 4 min

Fig. 8. LVDP chnages by the venom (25 µg) preincubated with atropine (100 µl) for 30 min

Discussion

In this study, in vitro negative inotropic, envenomation (Bay et al. 1997, Tavares et al. chronotropic and late arrhythmogenic effects 2004). Envenomation with H. lepturus which of H. lepturus envenomation in rats were is not painful is commonly seen in South shown with Langendorff apparatus. West Provinces of Iran and treated with in- The most interesting findings in envenomed tramuscular injection of Razi Institute poly patients by H. lepturus are hemoglobinuria, valent antivenom and followed by close hematuria, cutaneous necrotic ulcers and in monitoring. minority of cases disseminated intravascular In previous in vivo studies, bradycardia coagulation leading to death (Radmanesh 1990, (decreasing from 227 beats/min to 94 beats Shayesteh et al. 2011) as seen in Loxosceles /min) and ST elevation in lead II was in- J Arthropod-Borne Dis, K Pourkhalili et al.: Cardiotoxic and …

duced with intravenous injection of extreme as one of the most autonomic determinants doses (50 µg/kg) of venom in rabbits with no of cardiac contractility and coronary perfu- significant changes of lactate dehydrogenase sion (Gordon et al. 1998). In a parallel study, and Creatinine phosphokinase one hour after preinjection of propranolol (10 µl ) as a sym- envenomation showing no myocardial ne- pathetic antagonist had an additive role on crosis (Mirakabadi et al. 2010). In this paper, cardiotropic effects of H. lepturus venom a Langendorff model was assembled to an- supporting our hypothesis (Fig. 7). alyze the direct and indirect chronotropic Based on our study pre venom injection and inotropic effects of H. lepturus venom of atropine or incubated mixture of venom on rat hearts. with this drug, had significant neutralizing Our venom induced a persistant negative effect in contractility following envenomation dose response inotropic and chronotropic clearly showing that negative inotropic ef- effects in rat hearts until up to 60 min, similar to fect of this venom possibly was also depend- loxosceles envenomation (Shahbazzadeh et al. ent on cholinergic system by direct stimula- 2007, Chatzaki et al. 2012). This study pru- tion or activation of muscarinic receptors in dently approved that Hemiscorpius lepturus atria with releasing of acetylcholine from nerve cardiotoxicity was highly dependent on dose terminals like Tityus cerrulatus scorpion ven- and time of its administration. Coronary blood om (Teixeira et al. 2001). Post venom injec- flow was significantly decreased (10.4 ± 2.21 tion of Atropine had no neutralizing effect on to 3.16 ± 2.58 ) in envenomed hearts with no cardiotropic aspects possibly due to binding changes in LDH and CPK before and 60 min of the venom to its ion channel receptors after envenomation (Data not shown) ruling strongly. There is a great debate about the out the myocardial necrosis. According to efficacy of scorpion antivenom in neutraliza- the nature of poisonous animals, cardiotoxic tion of the detrimental effects induced by effects of scorpion venoms could be ex- envenomation (Gueron et al. 1967, Abroug plained with prevention or releasing of neu- et al. 1999, Ismail 2003, Chatzaki et al. 2012) rotransmitters including acetylcholine and since clinical efficacy of treatment for en- epinephrine from nerve terminals inducing venomed patients is affected deeply with the changes in heart rate and contractility time between scorpion sting and administra- (Tarasuik et al. 1994, Sauviat et al. 1995). tion of antivenom. Generally H. lepturus Our data suggest that H. lepturus venom venom like other poisonous animals have low could be suppressing, at least partially on the molecular weight diffusing rapidly to target adrenoceptors mimicking agent presenting in organs including kidney and heart while the venom or from varicosities in the heart like heavy chain molecules of antivenom have other poisonous creatutres (Gomes et al. not this ability making it unsuitable for 2010) in addition to direct binding to its treating envenomed patients (Ismail et al. myocardial receptors that their shape and 1983, Seyedian et al. 2010, Seyedian et al. origin will be clarified in further studies. In 2012). our experiment, pre and post injection of In our experiment, prevenom injection of epinephrine (10 µl) counteracted negative antivenom in addition to incubation of ven- inotropic and chronotropic effects induced om and antivenom completely antagonized with H. lepturus venom. It should be noted all deterimental effects including negative that coronary blood flow was not decreased inotropic, chronotropy, decreasing coronary by pre and post venom injection of adrena- blood flow and even late arrhythmogenic line (data not shown) indicating H. lepturus effects (bradycardia, QRS prolongation and indirect effect via supression of β receptors ST depression after 60 min) in isolated rat J Arthropod-Borne Dis, K Pourkhalili et al.: Cardiotoxic and … hearts while post venom injection had no Acknowledgements effect. It seems that antivenom could bind firmly to venom and prevents its direct and The authors would like to thank the au- indirect effects while injected before enven- thorities of Bushehr University of Medical omation but due to its high molecular weight Sciences for providing facilities. structure and different pharmacokinetic and biodistribution profile versus H. lepturus ven- References om had no treating effects in envenomed hearts even when injected 5 min after enven- Abroug F, Elatrous S, Nouria S, Haguiga H, omation (data not shown). Scorpion venoms Touzi N, Bouchoucha S (1999) generally act mainly on Na+ and to lesser de- Serotherapy in scorpion Envenomation: grees on calcium and potassium channels pro- a randomized controlled trial. Lancet. voking electrocardiographic changes (Gordon 354: 906–909. et al. 1998). Arrhythmogenic changes in our Bawaskar H, Bawaskar P (1998) Indian red envenomed hearts could be induced by late scorpion envenoming. Ind J Pediatr. ischemia or prolongation of repolarization 65: 383–391. phase with some purified toxins from H. Bey TA, Walter FG, Lober W, Schmidt J, lepturus as Hemicalcins and Heminecrolysin Spark R, Schlievert PM (1997) Lo- (Shahbazzadeh et al. 2007, Borchani et al. xosceles arizonica Bite Associated with 2011) acting on ion channels. Those effects Shock. Ann Emerg Med. 30: 701–703. were neutralized by prevenom injection of Razi Borchani L, Sassi A, Shahbazzadeh D, Strub Institute multivalent antivenom. Arrythmogenic JM, Tounsi-Guetteti H, Boubaker MS, changes with venom including QRS prolon- Akbari A, Van Dorsselaer A, El ayeb gation and ST depression were not observed M (2011) Heminecrolysin, the first during treatments with other drugs (atropine, hemolytic dermonecrotic toxin purified propranolol and adrenaline) and antivenom from scorpion venom. Toxicon. 58: 130– as control. 139. Chatzaki M, Horta C, Almeida M, Pereira N, Conclusion Mendes T, Dias-lopes C, Guimarães G, Moro L, Chávez-olórtegui C, Horta It seems that cardiotropic changes including M (2012) Cutaneous caused negative inotropism and chronotropism of H. by Loxosceles similis venom and neu- lepturus venom in addition to late tralization capacity of its specific arrhythmogenic manifestations has a detri- antivenom. Toxicon. 60: 21–30. mental effect in heart failure induced by en- Cupo P, Hering SE (2002) Cardiac troponin venomation in human beings. We believe that I release after severe scorpion enven- indirect and direct mechanisms are involved in oming by Tityus serrulatus. Toxicon. this phenomenon since injection of adrenaline 40: 823–830. and atropine according to their high and low Dehghani R, Fathi B (2012) Scorpion sting presence in the rat heart changed our results. in Iran: A review. Toxicon. 60: 919– Pre venom injection of Razi Institute 933. antivenom as the customary treatment could Gomes, HL, Andrich F, Mauad H, Sampaio neutralize its cardiotropic effects but unfor- KN, De lima ME, FIgueiredo SG, tunately it had no effect even 5 min after Moyses MR (2010) Cardiovascular ef- envenomations making its beneficial role on fects ofscorpionfish (Scorpaena plumieri) detrimental cardiac consequences questionable. venom. Toxicon. 55: 580–589. J Arthropod-Borne Dis, K Pourkhalili et al.: Cardiotoxic and …

Gordon D, Savarin P, Gurevitz M, Zinn- Perriere C (1995) Does crude venom justin S (1998) Functional anatomy of of the stonefish ( verrucosa) scorpion toxins affecting sodium chan- activate β-adrenoceptors in the frog nels. Toxin Rev. 17: 131–159. heart muscle? Toxicon. 33: 1207–1213. Gueron M, Stern J, Cohen W (1967) Severe Seyedian R, Jalali A, Babaee M, Pipelzadeh myocardial damage and heart failure in M, Rezaee S (2012) A biodistribution scorpion sting: Report of five cases. study of Hemiscorpius lepturus scor- Am J Card. 19: 719–726. pion venom and available polyclonal Ismail M (2003) Treatment of the scorpion antivenom in rats. J Ven Anim Toxins envenoming syndrome: 12-years ex- Trop Dis. 18: 375–383. perience with serotherapy. Int J Seyedian R, Pipelzadeh MH, Jalali A, Kim Antimicrob Agents. 21: 170–174. E, Lee H, Kang C, Cha M, Sohn ET, Ismail M, Shibl A, Morad A, Abdullah M Jung ES, RahmanI AH ,Mirakabady (1983) Pharmacokinetics of 125 I- AZ (2010) Enzymatic analysis of labelled antivenin to the venom from Hemiscorpius lepturus scorpion venom the scorpion Androctonus amoreuxi. using zymography and venom-specific Toxicon. 21: 47–56. antivenin. Toxicon. 56: 521–525. Jalali A, Pipelzadeh MH, Sayedian R, Shahbazzadeh D, Amirkhani A, Djadid ND, Rowan E (2010) A review of epide- Bigdeli S, Akbari A, Ahari H, Amini miological, clinical and in vitro physi- H, Dehghani R (2009) Epidemiologi- ological studies of envenomation by cal and clinical survey of scorpionism the scorpion Hemiscorpius lepturus in Khuzestan province, Iran (2003). (Hemiscorpiidae) in Iran. Toxicon. 55: Toxicon.53: 454–459. 173–179. Shahbazzadeh D, Srairi-abid N, Feng W, Latifi M, Tabatabai M (1979) Immunolog- Ram N, Borchani L, Ronjat M, Akbari ical studies on Iranian scorpion venom A, Pessah IN, De waard M, El ayeb M and antiserum. Toxicon. 17: 617–620. (2007) Hemicalcin, a new toxin from Mirakabadi A, Khatoonabadi S, Teimoorzadeh the Iranian scorpion Hemiscorpius S (2011) Antivenom injection time lepturus which is active on ryanodine- related effects of Hemiscorpius lepturus sensitive Ca2+ channels. Biochem J. scorpion envenomation in rabbits. Arch 404: 89–94. Razi Inst. 66: 139–145. Shapira MY, Haviv YS, Sviri S (1998) Mirakabadi A, Khatoonabadi S, Teimourzadeh Second degree atrio-ventricular block S, Sabiri GH (2010) Serum enzymes and cardiotoxicity secondary to enven- studies in scorpion (Hemiscorpius omation by the scorpion Leiurus lepturus) dose related envenomation in quinquestriatus (Yellow Scorpion')-an rabbits. Arch Razi Inst. 65: 83–89. indication for serotherapy? Hum Exp Prendini L (2000) Phylogeny and classifica- Toxicol. 17: 541–543. tion of the superfamily Scorpionoidea Shayesteh AA, Zamiri N, Peymani P, Latreille 1802 (, Scorpiones): Zargani FJ, Lankarani KB (2011) A an exemplar approach. Cladistics. 16: novel management method for dis- 1–78. seminated intravascular coagulation Radmanesh M (1990) Clinical study of like syndrome after a sting of Hemis- Hemiscorpion lepturus in Iran. J Trop corpius lepturus: a case series. Trop Med Hyg. 93: 327–332. Biomed. 28: 518–523. Sauviat MP, Garnier P, Goudey-perriere F, Srairi-abid, N, Shahbazzadeh D, Chatti I, J Arthropod-Borne Dis, K Pourkhalili et al.: Cardiotoxic and …

Mlayah-bellalouna S, Mejdoub H, Tavares F L, Sousa-e-silva MCC, Santoro M Borchani L, Benkhalifa R, Akbari A, L, Barbaro KC, Rebecchi IMM, Sano- El ayeb M (2008) Hemitoxin, the first martins IS (2004) Changes in hema- potassium channel toxin from the ven- tological, hemostatic and biochemical om of the Iranian scorpion Hemis- parameters induced experimentally in corpius lepturus. FEBS J. 275: 4641– rabbits by 4650. venom. Hum Exp Toxicol. 23: 477–486. Tarasiuk A, Sofer S, Huberfeld SI, Scharf Teixeira A, Fontoura B, Freire-maia L, SM (1994) Hemodynamic effects fol- Machado C, Camargos E, Teixeira M lowing injection of venom from the (2001) Evidence for a direct action of scorpion Leiurus quinquestriatus. J Tityus serrulatus scorpion venom on the Crit Care. 9: 134–140. cardiac muscle. Toxicon. 39: 703–709.