Introduction

Scorpion sting is one of the major health challenges for humans and animals, espe- cially in tropical and developing countries. More than 1,500 scorpion species have been identified worldwide (Lourenço, 2014; Lu- cas and Meier, 2017) and about 32 species were found in Iran, 12 of which have been detected in Khuzestan (Jalali and Rahim 2014; Rahimi et al., 2015).

The Buthidae are the largest family of scorpions and are found all over the world especially in tropical and semi-tropical re- gions, particularly the Middle East and Cen- tral Asia (Lourenço, 2014; Lucas and Meier, 2017). Mesobuthus eupeus is the most abun- dant species in Iran which is found in Khuz- estan province and is responsible for 21.7% of cases of scorpion sting in Khuzestan (Ra- himi et al., 2015; Dehghani et al.,  2017).

The venom of scorpions, especially the Buthidae family, consists of a variety of toxins and active biological compounds (Khoobdel et al., 2013; Erdeş et al., 2014; Laraba-Djebari et al., 2015) which can af- fect various tissues (Murthy and Zare, 2001; Murthy and Zare, 2002; Razi Jalali et al., 2015). Scorpion envenomation can lead  to the failure of many organs and major met- abolic and endocrine disorders through the release of large amounts of catecholamines, and vascular disorders (Petricevich,  2010). In some studies, scorpion stings were found to be associated with alterations in insulin, glucagon, and glucocorticoid secretion level, and in blood glucose levels (Murthy, 2000). However, there are few studies about the ef- fect of scorpion venom on thyroid secretion (Murthy and Zare, 1998).

To the best of the authors' knowledge,  there has been no documented data on the effects  of  M.  eupeus  venom  on  the endo-

crine system, including thyroid, endocrine pancreas and adrenal secretions. Since it is a native Iranian species, research into various aspects of the pathogenesis of its venom are important to improve diagnostic and thera- peutic methods. Therefore, the present study was designed to investigate the effects of Mesobutupus eupeus envenomation on plas- ma thyroid hormones (T3 and T4), insulin, glucagon, cortisol, and glucose levels in rats.

Materials and Methods

Scorpion venom

The venom of M. eupeus scorpion venom used in this study was provided by Razi Vac- cine and Serum Research Institute, Ahvaz which was prepared by electric shock and kept in lyophilized form. The concentration of crude venom protein was 815 mg/gven- om. Solutions of 0.05% and 0.1% of venom were prepared in 0.9% sodium chloride solu- tion in suitable volumes proportional to the number of rats in each envenomated group, as described below.

Laboratory animals

A  total  of  45  Albino  male  rats  (Wis- tar strain)  weighing  250–300  g aged  4 to   6 months were housed in groups of six, in plastic cages, in an air-conditioned room maintained at a temperature of 24 ± 2 °C and a relative humidity of 55 ±5%, with a 12-h light/12-h dark illumination cycle.  They were fed a commercial laboratory pellet diet and tap water ad libitum. All procedures were done in accordance with ethical guide- lines for care and use of laboratory animals, discarding of dead  animals  and  protection of the researcher against animal bites and were approved by the Experimental Animals Committee of Shahid  Chamran  University of Ahvaz, Iran.

Experimental design

Animals were randomly divided into 3 equal groups and were treated as  follows:

Group 1: (control group) 0.5 ml normal sa- line intraperitoneally (IP).

Group 2: M. eupeus  venom  at a dose  of  1 mg/kg body weight IP (0.5 ml of 0.05% solution for each rat).

Group 3: M. eupeus venom at a dose of 2 mg/kg body weight IP (0.5 ml of 0.1% solu- tion for each rat).

The doses of venom were selected based on previous studies by authors (Razi Jalali et al., 2015; 2016).

Blood collection

Sampling was performed at 8, 24, and 48  h after venom/saline injection; five rats from each group were sampled each time. Blood samples were collected with heparin via cardiac puncture following anesthesia with chloroform (Merck, Germany). After centrif- ugation the plasma was separated and stored at -20 ºC for subsequent measurements.

Plasma biochemical  analysis

The plasma levels of T4 and T3 were mea- sured using ELISA kits (Sigma-Aldrich). Cortisol, insulin and glucagon were also assessed  by  ELISA  (Monobind,  Germany)

according to the manufacturer's instructions. The amount of glucose was measured using colorimetric kits (Pars Azmun, Iran) via bio- chemical autoanalyzer (Biotecnica, Bt-1500, Italy).

Statistical analysis

Analysis of variance and Tukey post-hoc tests were employed to compare the data be- tween groups using SPSS software version  16 (SPSS Inc., Chicago, Illinois, USA). All values were expressed as mean ± standard error, and p < 0.05 was considered as statisti- cally significant.

Results

Clinical signs

The envenomated rats showed behavioral changes from aggression to depression. In addition, hemorrhage and hyperemia in the eyes and mucosal membranes were recorded in both intoxicated groups.

T3 measurement

The results of T3 evaluation showed that the levels of this hormone were significantly lower in group 2 than in the control group and in group 3 compared to the rest of the groups, at all sampling times (p <0.05) (Ta- ble 1). In addition, in group 3, the amount of

Table 1. Plasma triiodothyronine (T3) and thyroxine (T4) concentra- tion as mean ± SE in different groups and sampling times.

* Different uppercase letters in each column represent significant difference between groups.

** Different lowercase letters in each row represent significant difference between sampling times.

Group

Table 1. Plasma triiodothyronine (T3) and thyroxine (T4) concentra- tion as mean ± SE in different groups and sampling times.

Insulin (µIU/dl)                                                      Glucagon (ng/ml)

8 h                     24 h                    48 h                     8 h                     24 h                    48 h

Group 1 (control)

Group 2 (venom 1 mg/kg)

Group 3 (venom 2 mg/kg)

12.70 ± 0.92

A* 8.40 ± 1.08

B 8.13 ± 0.74

B

12.13 ± 1.81

A 9.36 ± 1.29

B 7.06 ± 1.29

B

11.40 ± 1.92

A 10.60 ± 1.20

B 8.84 ± 1.11

B

123.67 ± 2.60

A* 149.20 ± 2.70

Ba** 156.50 ± 2.46

Ca

122.25 ± 2.27

A 154.40 ± 0.67

Bb 168.80 ± 1.02

Cb

121.50 ± 1.19

A 139.80 ± 1.70

Ac 136.00 ± 1.81

Ac

* Different uppercase letters in each column represent significant difference between groups.

** Different lowercase letters in each row represent significant difference between sampling times.

T3 significantly increased over time, so that the highest hormone level was at 48 h post injection (p <0.05).

T4 measurement

Based on the results of T4 measurement (Table 1), the hormone levels decreased in both envenomated groups  (groups  2 and 3) in comparison with the control group, so that the difference was significant at 8 and 24 h (p <0.05). However, with the passage of time at 48 h after venom injection, this decrease was moderated and there was no significant difference between groups at any of the sam- pling times.

Insulin measurement

Based on the results (Table 2), there was a significant decrease in insulin level in the in- toxicated groups (groups 2 and 3) compared to the control group at all sampling times (p<0.05).

Glucagon measurement

The administration of venom signifi- cantly increased glucagon level in group 2 compared to the control group and in group  3 compared to the previous two groups at 8 and 24 h post injection (p <0.05) (Table 2). Additionally, in both intoxicated groups, the maximum amount of hormone was observed at 24 h post injection (p <0.05).

Cortisol measurement

A significant increase in cortisol concen- tration was noted in both groups receiving the venom (groups 2 and 3) compared to the control group at 8 and 24 h following en- venomation (p<0.05) (Table 3). However, in both mentioned groups, the level of cortisol decreased significantly at 48 h in compari- son to the previous sampling times (p <0.05).

Glucose measurement

Glucose levels in both venom injected groups (groups 2 and 3) were significantly increased compared to the control group at  all sampling times (p <0.05) (Table 3). How- ever, there was no significant difference be- tween two envenomed groups in terms of glucose levels.

Discussion

In the present study, injection of M. eupeus venom resulted  in  a  significant  reduction  in the thyroid hormones (T3 and T4). The highest decrease was observed in the third group (high dose venom) at 8 h after injec- tion, considering the half-life of T3 which is only 1 day while the half-life of T4 is 5-7 days. These changes might be attributable to an autonomic storm through the release of catecholamines and angiotensin II  following

Table 1. Plasma triiodothyronine (T3) and thyroxine (T4) concentra- tion as mean ± SE in different groups and sampling times.

* Different uppercase letters in each column represent significant difference between groups.

** Different lowercase letters in each row represent significant difference between sampling times.

envenomation, which led to a decrease in in- sulin secretion and subsequent reduction of T3 and T4 concentration in the blood. These findings are consistent with other previous studies in this regard. In a study performed by Murthy and Zare (1998) a significant decrease in T4 and T3 levels was observed following the injection of Mesobuthus ta- mulus venom in dogs. They also obtained similar results regarding  T3  concentration in rabbits. In addition, while anti-venom ad- ministration resulted in an increase in serum T3 level in dogs, it could not prevent venom induced hormonal changes in rabbits. How- ever, the intracerebroventricular injection of Kaliotoxin (KTX) fraction of Androctonus australis venom, in another  study,  resulted in the stimulation of neuro-endocrine re- sponse with significant rise in serum T3, T4, and TSH levels at 24 h post-injection in rats which was associated with inflammatory cell infiltration and imbalanced redox status in both hypothalamus and thyroid tissue (Lad- jel-Mendil et al., 2016). This diversity in the results might be due to the difference in the type of studied scorpion species and venom as well as the route of venom administration. According to the results of this study, glu- cagon levels were significantly increased   in

the venom treated groups at 8 and 24 h after injection in a dose-response manner. How- ever, the levels of this hormone returned to normal in both groups at 48 h after enven- omation. This effect is probably mediated through the adrenergic stimulation of endo- crine pancreas as well as other tissues. Sim- ilar alterations in glucagon secretion were also reported by Johnson and Ensinck (1976) following pancreas perfusion with Leiurus quinquestriatus scorpion toxin in rats. This reduction in glucagon secretion was associat- ed with the stimulation of sympathetic nerve terminals in pancreas tissue and increased norepinephrine release which was success- fully prevented in the presence of alpha and beta-adrenergic blocking agents in rat. In ad- dition, subcutaneous administration of Me- sobuthus tamulus scorpion venom resulted in an increased blood glucagon in dogs. These hormonal changes seem to result in an im- balance in the carbohydrate metabolism and the inability of vital organs to use metabolic substrates (Murthy and Haghnazari,  1999).

The present study revealed that there was  a significant decrease in insulin levels in the envenomated rats compared to the control group, with the highest reduction at 8 and 24 h after  venominjection.  In other experimen-

tal studies, hypoinsulinemia or insulin resis- tance was reported following  the  injection of various scorpion spp. venom. During the study conducted by Murthy and Haghnazari (1999) and Zare et al. (1994), the same al- terations in insulin level was recorded fol- lowing Mesobuthus tamulus venom admin- istration in dogs. Furthermore, Androctonus australis hector scorpion toxin was associat- ed with severe inflammatory response, liver tissue damage and hyperglycemia accompa- nied by hyperinsulinemia, indicative of insu- lin resistance in a previous study in rats. It is also possible that the insulin measured after venom injection was not functionally active. Moreover, pretreatment with cytokine antag- onists, in the same study, significantly de- creased inflammatory biomarkers and plas- ma glucose levels as well as hepatic tissue damage and metabolic disorders which was suggestive of the crucial role of inflammato- ry cytokines (IL-6 and TNF-α) in the com- plications induced by the scorpion venom (Taibi- Djennah and Laraba-Djebari,  2015).

In addition, exogenous insulin admin- istration was demonstrated to reverse he- modynamic, cardiovascular, metabolic, electro-cardiographic (ECG) changes and pulmonary oedema in the experimental ani- mals and in the scorpion sting victims which represents the key role of insulin suppres- sion in the pathogenesis of the toxin and also suggests that the insulin receptor and the signaling pathways are not defective (Mur- thy, 2013, 2014a; Murthy et al., 1988, 1990, 1991; Yugandhar et al., 1999). It appears that scorpion envenomation, including M. eu- peus, results in a substantial release of cat- echolamines and Angiotensin II which can inhibit glucose-induced secretion of insulin from the beta cells of islets in the endocrine pancreas as well as antagonizing the  actions

of insulin by promoting glycogenolysis which leads to hyperglycemia. The suppres- sion of insulin secretion or insulin resistance along with a rise in catabolic counter-regu- latory hormones including glucagon, corti- sol and glucose level might be responsible for the pathogenesis of a variety of clinical symptoms. Consequently, M. eupeus venom, like many other scorpion species, can induce a syndrome of energy  scarcity  and inabili-  ty of vital organs to consume the metabolic substrates causing multi-organ system fail- ure (Murthy, 2014b).

In the current experiment, the injection of scorpion venom in both groups led to a sig- nificant increase in cortisol levels. The high- est hormone concentration was observed at 8 h after receiving the toxin and decreased over time, so that cortisol changes were not sig- nificant within 48 h. Other previous studies have found similar findings. Subcutaneous administration of M. tamulus resulted in an increased blood cortisol in dogs and rabbits (Murthy and Haghnazari, 1999; Zare et al., 1994). Excessive release of catecholamines following scorpion venom intoxication is, once again, likely responsible for increased endogenous glucocorticoids which,  in turn, is often associated with carbohydrate intol- erance (Murthy, 2014b).

The results of glucose measurements  in the present study showed a significant in- crease in the level of this analyte after in- jection of M. eupeus venom at all sampling times compared to control group. Consider- ing that there was no significant difference between the two groups of venom recipi- ents, it seems that changes in glucose do not correlate with the dose of injected venom. Hyperglycemia has already been observed following injection of various scorpion spp. toxins including Mesobuthus tumulus   (Mur-