Toxicon 55 (2010) 11251131

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Toxicon
journal homepage: www.elsevier.com/locate/toxicon

Biochemical prole of dogs experimentally envenomed with Tityus serrulatus scorpion venom
` E.L. Ribeiro a, M.C.L. Pinto a, C.R. Labarrere a, P.R.O. Paes a, F.O. Paes-Leme a, vez-Olortegui b, M.M. Melo a, * C. Cha
a b

Escola de Veterinaria, Universidade Federal de Minas Gerais, Minas Gerais, 30123-970 Belo Horizonte, MG, Brazil Departamento de Bioqumica-Imunologia, Instituto de Ciencia Biologicas, Universidade Federal de Minas Gerais, Minas Gerais, 30123-970 Belo Horizonte, MG, Brazil

a r t i c l e i n f o
Article history: Received 6 July 2009 Received in revised form 2 October 2009 Accepted 2 January 2010 Available online 7 January 2010 Keywords: Tityus serrulatus Envenomation Blood analysis Urine analysis Dog

a b s t r a c t
The aim of this study was to evaluate the canine blood and urinary proles after envenomation by Tityus serrulatus venom. Twelve dogs were randomly distributed into two equal groups. Control group animals received 0.5 mL phosphate buffered saline (PBS) injected subcutaneously into the internal portion of the left thigh, whilst dogs in the envenomed group were injected with scorpion venom (250 mg/kg in 0.5 mL PBS). No signicant alterations were detected in the urine of envenomed dogs. Levels of plasma glucose and serum urea, creatinine, total protein, potassium, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase (LDH), and amylase were determined. Semi-quantitative analysis of serum cardiac troponin I (cTnI) was performed using an immunochromatographic test. The concentrations of cortisol and insulin were determined using commercial radioimmunoassay kits. Increases in serum cortisol levels in experimental group animals coincided with hyperglycaemia and was probably a response to pain. Increased insulin levels were observed during the hyperglycaemic peaks. Envenomed dogs presented discreet increases in ALT, AST and CK, but no alterations in LDH, amylase, cTnI, urea, creatinine and potassium levels were observed. It was concluded that the venom of T. serrulatus induces blood and urinary biochemical changes in dogs. 2010 Elsevier Ltd. All rights reserved.

1. Introduction Scorpion stings represent an important and serious public health problem in certain regions of Brazil and in other parts of the world. In Brazil, approximately 10,000 human cases of scorpion sting are treated at hospital centres and notied annually, and 50% of these cases occur in Minas Gerais and Sao Paulo states. The scorpion species Tityus serrulatus is the most prevalent (95%) and accounts

* Corresponding author. Tel.: 55 31 34092229; fax: 55 31 34092230. E-mail addresses: marilia@vet.ufmg.br, marilia.melo@pq.cnpq.br (M.M. Melo). 0041-0101/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2010.01.001

for fatal stings, especially among children (Soares et al., 2002; Cupo et al., 2003). Scorpion venom comprises a complex mixture of short and long chain peptides associated with mucopolysaccharides and small amounts of hyaluronidases and neurotoxins (Gazarian et al., 2005). The mechanism of action of the toxins involves blockage of the ion channels of the neuronal membranes and release of neurotransmitters from the postganglionic nerve terminals. Some toxins stimulate the entry of sodium through the voltage- and non-voltage dependent sodium channels, whilst others obstruct the potassium channels and impede the return of this ion to the cell, thus causing its accumulation in the extra cellular medium. In either case, the result of such action is

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membrane depolarisation followed by an action potential and the massive release of neurotransmitters, in particular acetylcholine and noradrenaline from the postganglionic nerve terminals, and adrenaline from the adrenal medulla (Ismail, 1995). The effects can be adrenergic or cholinergic depending on the neurotransmitter and the type of neuron, and the symptoms may range from excruciating pain at the site of the sting to grave systemic effects (Cordeiro et al., 2006; Ribeiro et al., 2009). Cardiovascular and haemodynamics alterations (congestive heart failure, pulmonary oedema and arterial hypertension) are the most severe consequences in this envenomation, usually followed by cardiac muscular lesions (Cupo et al., 2003, 2007). The occurrence of myocarditis after scorpion envenomation might be multifactorial, being caused by cardiotoxic effect of catecholamines (i.e. hypoxia), vasoconstriction and high oxygen demand, lactic acid accumulation (Raab, 1960; Murthy and Hase, 1994; Ismail, 1995; Sofer et al., 1996; Meki and ElDean, 1998; Meki et al., 2003); by direct venom effect (Teixeira et al., 2001) and by cytokines (Magalhaes et al., 1999). The cardiac troponin I (cTnI) concentration is highly correlated with the occurrence of alterations in electro and echocardiogram features and CK-MB and AST increase. The cTnI have been observed in moderate and serious scorpionism cases, in which is observed a 50-time increase compared to the normal value (Bucaretchi et al., 1995; Cupo and Hering, 2002; Cupo et al., 1994, 2003). Some metabolic disturbs are reported such as, hypersecretion and hypermotility of the digestive tract, causing excessive salivation, nausea, vomiting, abdominal pain, electrolytic imbalance and haemoconcentration, which leads to body dehydration (Bertazzi et al., 2003; Ribeiro et al., 2009). Pancreatitis caused by exocrine hypersecretion might be the result of the direct action of neurotoxins on pancreatic cells, and also, by the neurogenic activity affected by cholinergic hyperstimulation (Sankaran et al., 1983; Possani et al., 1992; Bucaretchi et al., 1995; Fletcher et al., 1996; Cupo et al., 2003). Some researchers (Fukuhara et al., 2003, 2004) have reported a correlation of the levels of kinins (bradykinin and kallikrein) with the production of cytokines (IL-1, IL-6 and TNF-a) and stimulation of the hepatic synthesis of acute-phase proteins in human patients. Changes in cortisol and insulin levels observed in scorpion accidents are associated with the discharge of catecholamines and the stress produced by pain. One example of increased cortisol, accompanied of hyperglycaemia, was reported in a study involving canine inoculation with Mesobuthus tamulus concanesis venom (Murthy and Haghnazari, 1999). Ismail and Abd-Elsalam (1988) have stated that hyperglycaemia is caused by pancreatitis followed by insulin production and secretion decrease. Inhibition of insulin release and stimulation of glucagon secretion, directly or not, are often observed in cases of scorpionism. Such effects, gather with vasoconstriction, hypoxia and increased energy consumption, constitute the Energy Decit Syndrome, the most severe outcome of scorpion envenomation (El-Asmar, 1984; Ismail and AbdElsalam, 1988; Murthy and Hase, 1994; Yugandhar et al., 1999).

Although dogs have been used in studies about physiological and pharmacological actions of scorpion venoms, especially for measuring cardiovascular parameters (Bartholomew et al., 1977, Murthy and Haghnazari, 1999; Tarasiuk and Sofer, 1999; Cordeiro et al., 2006), researches concerning a complete biochemical prole, after experimentally envenomation by T. serrulatus, are rare. In the present report we investigated the T. serrulatus venom effects in cardiac, pancreatic, hepatic, renal and metabolic functions in dogs. 2. Material and methods This research was approved on 12th June 2006 by the Ethical Committee for Animal Experimentation of the Universidade Federal de Minas Gerais (UFMG), under the protocol number 15/2006. 2.1. Animals and venom The study population comprised 12 healthy (as determined by clinical and laboratory tests) adult male mongrel dogs (average weight 14.2 5.4 kg) obtained from the Centres for Control of Zoonosis of the municipalities of Belo Horizonte and Betim, MG, Brazil. The experiment followed a random design with subdivided plots. Animals were distributed randomly into two equal groups. Those in the control group (n 6) received 0.5 mL of phosphate buffered saline (PBS) injected subcutaneously into the internal portion of the left thigh. Dogs of the envenomed group (n 6) were injected in a like manner with T. serrulatus venom (250 mg/kg ressuspended in 0.5 mL PBS) from a lyophilised pool originating from various scorpion specimens supplied by the Laboratory of Immunology and Biochemistry from Instituto de Ciencias Biologicas (ICB) UFMG. The protein venom concentration was determined by using the Folin-Phenol Ciocalteau reagent (Lowry et al., 1951). The venom dose inoculation in was performed in a pilot experiment. 2.2. Samples and biochemical analyses Urine samples were collected prior to the experiment and at 8, 24 and 48 h after venom inoculation using a urethral catheter. The density of each sample was determined using a urinometer (Ningbo Utech International CO LTDA, China), whilst pH, albumin, glucose, urobilinogen, bile salts and haemoglobin were assessed with the aid of reaction dipsticks (Uri-Test 10 Inlab, Alemanha). Following centrifugation, the urine sediment was analysed under the optical microscope. Blood samples were collected prior to the experiment and at 2, 6, 12, 24, 48 and 72 h after treatments by cephalic vein puncture, in tubes with anticoagulant sodium uoride to obtain plasma to glucose measurements, and without anticoagulant, to collect serum and achievement biochemical proles, either analysed immediately. Plasma glucose and serum urea, creatinine, total protein, potassium, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase (LDH), and amylase were determined with

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the aid of commercial kits from Synermed (Westeld, IN, USA) and a Cobas Mira Classic chemical analyser (Global Medical Instrumentations, Ramsey, MN, EUA). The semiquantitative analysis of serum cardiac troponin I (cTnI) was performed using the immunochromatographic Hexagon Troponin Plus; (Human International, Wiesbaden, Germany) test in order to detect concentrations above 1 ng/ mL. The concentrations of cortisol and insulin were determined using commercial radioimmunoassay kits (Coat-A Count; Diagnostic Products Corporation, Los Angeles, CA, USA) and a Cobra II Auto-Gamma counter (Packard Instrument Co. Meriden, CT, USA).

2.3. Statistical analysis Quantitative data were expressed as mean SD. Normal distributed data (urea, amylase, potassium, glucose, creatinine, total protein, AST, ALT, CK, LDH, cortisol and insulin) were subjected to analysis of variance and the mean values (which showed interaction signicant group time) were compared using the post-hoc Tukey test (P < 0.05). For variables that presented a non-parametric distribution (urinalysis), Friedman and Mann-Whitney tests were applied to compare times and groups, respectively (Sampaio, 2007). Statistical analyses were carried out with the aid of SAS (Cary, NC, USA) and Sistema para Analises Esta tsticas e Geneticas (Fundaao Arthur Bernardes, Viosa, MG, Brazil) software. 3. Results and discussion This canine envenomation was conducted after the determination of an optimal dose of venom, in a pilot experiment. The parameters evaluated consist of clinical examination, measurement of systolic blood pressure, electrocardiogram and echocardiography (unpublished data) and blood prole (Ribeiro et al., 2009). The rst dose (25 mg/kg) administrated in 12-pound dog, mimicked the amount of venom inoculated in a natural accident (0.25 0.4 mg) (Hassan, 1984). The second (50 mg/kg) and third doses (250 mg/kg) were used in order to cause a clinical picture described in literature. Cordeiro et al. (2006) made use of the same dose as ours (250 mg/kg) reporting leukocytosis, policitemia, electrocardiograph alterations and CK-MB elevation.

Urine samples collected at various evaluation times from animals within the control and treatment groups presented no signicant differences with respect to colour, aspect or odour. However, urine density varied in the range 1.0471.055 within the control group and between 1.050 and 1.059 within the envenomed group (P < 0.05). Although these values exceeded the upper limit for canines (1.0151.045) (Stockham and Scott, 2002), such variation does not indicate that the kidneys were incapable of concentrating urine following envenomation, since the densities were elevated in both groups even prior to the experiment. The pH values of the urine of animals within the envenomed group were slightly lower (6.06.8) than those of members of the control group (6.87.1). It is known that scorpionism produces either acidosis caused by vasoconstriction and tissue hypoxia (Murthy and Hase, 1994) or alkalosis as a result of H loss (vomiting) and hyperventilation (dyspnoea and tachypnea) (El-Asmar, 1984; Mag alhaes et al., 2000; Andrade et al., 2004). Hence, the observed alterations in urine pH may reect the effect of venom on the acid-base balance and the subsequent response of the organism via compensation mechanisms. One dog, from envenomed group, showed glycosuria after 8 h. The urine was submitted to the determination of glucose by the same method described for determining the plasma concentration and showed 191 mg/dl. Glycosuria occurs when the capacity of reabsorption of renal tubules is exceeded, either by increases in blood glucose (greater than 180 mg/dL), or by injury or renal diseases that increase or decrease the lter absorption (Finco, 1997). Abnormal alterations in the parameters associated with urinalysis have rarely been detected following scorpion poisoning, except for severe clinical cases exhibiting glucosuria and hematuria. The results of urinalysis, including microscopic analysis of sediment, indicated that the renal function of dogs remained normal after envenomation. There were no signicant differences (P < 0.05) between the two groups regarding the levels of serum urea and creatinine (Table 1), conrming the results of urinalysis. Pharmacokinetic studies in rats have previously shown that scorpion venom spreads rapidly from blood to tissues, and especially the kidneys, reaching a maximum concentration around 15 min after inoculation (Ismail and Abd-Elsalam, 1988; Santana et al., 1996; Nunan et al., 2003, 2004). Furthermore, there are reports of the occurrence of acute renal

Table 1 Urea, creatinine, total protein (mg/dl) and potassium (mmol/L) serum levels in dogs after placebo (control) and 250 mg/kg T. serrulatus venom inoculation (envenomed) in different times. Time (h) Urea (mg/dL) Control 0h 2h 6h 12 h 24 h 48 h 72 h 39.2 10.5 33.8 2.4 34.3 6.3 37.3 10.0 34.0 7.4 28.7 3.8 38.0 7.5 Envenomed 31.3 9.6 33.3 10.4 33.3 8.6 29.5 9.4 25.0 10.8 30.8 9.8 28.7 8.9 Creatinine (mg/dL) Control 1.03 0.25 0.90 0.27 0.90 0.32 0.97 0.25 0.88 0.26 0.82 0.24 1.05 0.42 Envenomed 0.85 0.45 0.87 0.43 0.80 0.37 0.82 0.40 0.65 0.25 0.78 0.20 0.77 0.15 Total protein (mg/dL) Control 6.22 0.75 5.95 0.83 6.32 0.76 6.20 0.70 6.10 0.72 5.82 0.30 6.23 0.77 Envenomed 7.08 0.95 7.10 1.06 7.22 1.17 7.12 0.94 6.95 0.91 6.90 1.56 6.97 1.47 Potassium (mmol/L) Control 3.84 1.38 4.46 0.89 4.87 1.37 4.36 0.64 4.27 0.83 4.14 0.60 4.36 0.65 Envenomed 2.86 0.64 2.68 1.10 2.97 0.93 3.20 0.98 3.59 1.38 2.75 0.30 2.83 0.61

Mean values ( standard error) are shown for the six animals in each group. There was no signicant difference at P > 0.05 according to Tukey test in groups and times.

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failure, with increased urea and uric acid concentration, diminished urinary volume and decreased creatinine secretion, in patients that had been stung by scorpions of the genera Androctonus, Leiurus and Buthus (Ismail and Abd-Elsalam, 1988). Since the excretion and serum concentrations of urea and creatinine were normal within the envenomed group, it is clear that T. serrulatus venom in the doses used did not cause alterations in the renal function dogs. There were no differences between control and envenomed animals regarding total serum protein content (Table 1), and all values remained within the normal limits (5.47.7 g/dL, Jain, 1993). These results not only conrm that the renal function was normal but also indicate the absence of dehydration. Four dogs from the envenomed group presented sialorrhoea within 5 h after inoculation of venom, and two dogs exhibited abdominal pain on palpation and vomiting (within 1 and 3.5 h, respectively). Such symptoms indicated hypersecretion and increased motility of the intestinal tract, resulting from cholinergic stimulation of the parasympathetic system (Ismail, 1995). Earlier reports (Bucaretchi et al., 1995; Cupo et al., 1994, 2003) have described loss of potassium owing to vomiting, sialorrhoea and diarrhoea in humans poisoned by scorpions. Although such symptoms were observed in the studied dogs, there were no alterations in potassium serum concentrations (Table 1). Amylase activity remained normal (3002000 U/L) (Jain, 1993) for animals of both groups, with the exception of a signicant variation (P < 0.05) in the values observed between 24 and 72 h (Table 2) in dogs of the envenomed group, probably because the amylase limits are wide. Although a secretagogue effect of scorpion venom has been observed in pancreatic acinar cells in vitro (Fletcher et al., 1996), the present results relating to amylase activity demonstrated that the parasympathetic stimuli did not inuence pancreatic acini and, consequently, exocrine discharge remained stable. In contrast, stomach function was inuenced by parasympathetic stimuli as shown by the responses (i.e. increased gastric secretion and vomiting) presented by two experimental dogs. Normality of renal function was further conrmed by the absence of increased amylase activity in experimental dogs. If glomerular ltration had been diminished by the action of scorpion venom, increased activity of the long-lasting amylase would have been observed during the experimental period.

Increases of 60 and 75% in the activities of ALT, in comparison with pre-treatment levels, were observed in animals of the envenomed group at 2 and 6 h after envenomation, therefore, without statistic difference (Table 3). In two envenomed dogs, ALT activity was very high at 2 h (115 and 122 U/L) and 6 h (178 and 185 U/L), and in one of these dogs ALT activity remained high even at 12 h (149 U/ L) and 24 h (125 U/L) after venom administration. Abdominal pain on palpation, as mentioned previously, may be, possibly, caused by hepatic lesions. Pharmacokinetic studies (Nunan et al., 2003, 2004) of the distribution of T. serrulatus venom in rats have shown that the concentration of toxins in the liver reached their highest level some 15 min after inoculation. However, there are no reports concerning the hepatotoxic activity of this scorpion venom or its effect on liver ion channels. Hepatic toxicity in scorpion accidents is rare, and there are only few descriptions of accidents by Hemiscorpious lepturus scorpions (Pipelzadeh et al., 2006). Also, they are rare reports in the medical literature regarding ALT activity in envenomed humans. In dogs, ALT is the most important hepatic enzyme, and in this research, 250 mg/kg of T. serrulatus venom caused liver toxicity. It is important to note that in experiment pilot (25 mg/kg), the ALT concentrations stayed within normality. The troponin complex consists of troponin C (containing Ca-binding sites), troponin T (which interacts with tropomyosin laments) and troponin I (an inhibitory component). All three subunits participate in the interaction among actin, myosin and tropomyosin resulting in the contraction of the muscle bre (Cardinet, 1997). Although troponin I is present in both cardiac and skeletal muscles, the human cardiac isoform is coded by a specic gene, and presents a particular amino acid sequence. Hence, immunological tests that employ antibodies for specic epitopes are very useful in the diagnosis of myocardial lesions (Bodor et al., 1992; Cupo and Hering, 2002; Meki et al., 2003; Diniz et al., 2007), particularly those that are undetectable by conventional enzymatic methods. The high sensitivity and specicity of such immunological tests, associated with complementary tests (i.e. AST, LDH-1, CKMB and myoglobin evaluation), have shown consistent results when cTnI concentrations are elevated. In the present study, the cTn1 levels of all animals tested (independent of group) were below the detection limit of 1 ng/mL. Considering that the normal cTnI concentration in

Table 2 LDH, CK and amylase (U/L) serum levels in dogs after placebo (control) and 250 mg/kg T. serrulatus venom inoculation (envenomed) in different times. Time (h) LDH (U/L) Control 0h 2h 6h 12 h 24 h 48 h 72 h 152.2 133.4 128.2 101.7 73.2 40.3 103.8 60.6 103.5 119.9 84.0 49.7 98.7 63.6 Envenomed 60.7 36.8 90.5 28.7 75.8 39.1 105.0 78.1 106.5 65.9 64.2 29.5 90.8 67.6 CK (U/L) Control 212.0 94.6 203.5 117.4 156.7 100.6 154.7 54.1 163.0 113.0 102.7 62.0 159.0 128.1 Envenomed 79.2 42.7* 139.5 115.7 130.0 91.8 140.3 57.6 151.2 86.6 87.3 26.3 103.2 37.7 Amylase (U/L) Control 776.5 48.6 763.2 76.7 816.2 65.1 800.7 116.1 848.2 164.6 829.2 116.1 866.7 161.3 Envenomed 894.8 188.1 899.0 152.1 891.8 172.8 793.0 206.8 700.7 155.5 782.3 216.7 910.3 290.4**

Mean values (standard error) are shown for the six animals in each group. There was signicant difference at P < 0.05 according to Tukey test *when compared, **when compared with time (24h).

E.L. Ribeiro et al. / Toxicon 55 (2010) 11251131 Table 3 ALT and AST (U/l) serum levels in dogs after placebo (control) and 250 mg/ kg T. serrulatus venom inoculation (envenomed) in different times. Time (h) ALT (U/L) Control 0 2 6 12 24 48 72 55.0 25.2 50.3 15.1 57.5 27.5 55.2 19.8 43.8 15.2 47.8 20.2 46.7 23.5 Envenomed 51.7 17.5 83.2 56.37 89.7 55.65 70.3 43.6 60.8 34.1 56.8 30.2 51.7 22.5 AST(U/L) Control 59.3 17.8 51.0 11.4 55.7 12.9 53.8 17.6 50.0 8.1 47.5 9.8 50.0 9.8 Envenomed 35.5 6.8 57.0 22.2* 61.8 16.4* 46.7 13.2 35.5 9.1 32.2 5.1 36.0 6.1

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Mean values (standard error) are shown for the six animals in each group. There was signicant difference at P < 0.05 according to Tukey test *when compared with control group at the same time.

dogs varies between 0.03 and 0.07 ng/mL (Sleeper et al., 2001), the results demonstrated that no myocardial lesions were induced in envenomed animals. There were no alterations in CK, LDH and cTnI, which were done to evaluated cardiac lesions (Table 2). Before T. serrulatus venom inoculation (time 0 h), it was observed difference within groups, but it is important to note that all values are within normal values (20473 U/L) (Kaneko et al., 1997). Since CK is present in the myocytes and plays a role in muscle contraction, this enzyme represents a sensitive and short-lived marker of muscle lesion. However, only large rises in CK levels present clinical signicance since intramuscular injections, wounds and prolonged decubitus can cause increases in CK activity (Cardinet, 1997; Kramer and Hoffmann, 1997). In humans, changes in the levels of CK isoenzyme MB have been correlated with cardiac lesions caused by myocardial infarction (Hetland and Dickstein, 1998; Ooi et al., 2000), and scorpion envenoming syndrome (Cupo and Hering, 2002; Cupo et al., 2003; Meki et al., 2003). Although there are dissimilarities regarding the tissue distribution of CK isoenzymes in domestic mammals, different CK isoforms can be found in the intestine, spleen and lungs of dogs. However, unlike the case for humans, dog CK-MB is not a reliable indicator of cardiac lesion (Wyatt et al., 1998). In the present study, large discrepancies in total CK and CK-MB levels were found within the experimental group, hence CK-MB activity was not taken into consideration (unpublished data).

In envenomed animals, AST activity (Table 3) increased signicantly (P < 0.05) at 2 and 6 h after venom inoculation, but returned to normal at 24 h. Since these dogs showed no increases in LDH and CK levels, the elevated AST levels observed could be explained on the basis of hepatic lesions and the consequent release of this enzyme, as ALT activity increases signicantly at same time (2 and 6 h after venom inoculation). Considering plasma glucose levels (Table 4), there were, in general, no signicant differences (P > 0.05) between control and envenomed animals or between samples collected at the various evaluation times. At 2 and 6 h after venom inoculation, the glucose mean values were elevated above to superior limit to dogs (60110 mg/dL) related by (Jain, 1993) with high standard deviations, including the dog that had glycosuria. With respect to cortisol levels (Table 4), the statistical difference between groups was observed at 2 h, which the envenomed group presented the highest mean value (9.08 mg/dL). This value was statistically different from that of the control group and exceeded the normal limits for dogs (0.966.81 mg/dL) (Kaneko et al., 1997). Subsequently the levels of cortisol tended to decrease. There were no signicant differences (P > 0.05) in the levels of insulin (Table 4) between the two groups of animals at any of the evaluation times (Table 3). The mean values for all animals, independent of group, were below the normal limits for dogs (520 mU/mL) (Kaneko et al., 1997). However, it is notable that the highest values of insulin in the envenomed group were identied in the times when glucose was higher. Considering the envenomed dogs individually, the serum insulin levels of three dogs increased between 2 and 12 h after inoculation to 7.5, 14.0 and 15.0 mU/ml (highest values in this period), an effect that was concomitant with an increase in your plasma glucose concentration (109, 128 and 292 mg/dl). After these results, possibly cortisol elevation was the cause of hyperglycemia described. It has been reported (Cunningham, 2004) that glucocorticoid response is proportional to the severity of stress, i.e., the lower the stress the lower the production of cortisol. In human accidents caused by scorpions, the local pain is the clinical sign more describe (Cupo et al., 1994; Bucaretchi et al., 1995) and also, in rare relates of animals pet (Cardoso et al., 2004; Ribeiro et al., 2009). In this work, the dogs showed acute pain for 6 h, in agreement with the results of cortisol.

Table 4 Blood plasma glucose, serum cortisol and insulin levels in dogs after placebo (control) and 250 mg/kg T. serrulatus venom inoculation (envenomed) in different times. Time (h) Glicose (mg/dl) Control 0 2 6 12 24 48 72 87.0 8.7 85.8 5.8 81.1 15.9 80.5 12.3 84.8 10.2 85.1 11.2 82.0 6.8 Envenomed 83.6 11.7 130.8 81.1 116.1 28.8 81.6 7.0 78.6 9.2 83.5 9.6 85.0 11.2 Cortisol (ng/dl) Control 3.45 1.45 2.35 0.85 2.67 1.08 3.03 1.42 2.32 1.40 3.08 0.77 2.75 1.48 Envenomed 3.03 1.32 9.08 9.78* 5.87 6.61 3.57 2.03 3.85 2.30 2.38 0.57** 2.58 0.49 Insulina (mU/mL) Control 5.57 5.83 6.00 4.21 4.30 1.77 7.90 10.38 2.23 1.40 3.57 2.28 3.22 0.86 Envenomed 3.55 1.69 6.65 4.11 7.72 5.12 5.12 2.73 3.95 2.78 4.53 4.85 4.45 1.71

Mean values (standard error) are shown for the six animals in each group. There was signicant difference at P < 0.05 according to Tukey test *when compared with control group at the same time, and **when compared with time (2h).

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E.L. Ribeiro et al. / Toxicon 55 (2010) 11251131 Cardinet, G.H., 1997. Skeletal muscle function. In: Kaneko, J.J., Harvey, J.W., Bruss, M.L. (Eds.), Clinical Biochemistry of Domestic Animals, fth ed. Academic Press, San Diego, pp. 407440. Cordeiro, F.F., Sakate, M., Fernades, V., Cuyumjian, P.R., 2006. Clinical and cardiovascular alterations produced by scorpions envenomation in dogs. J. Venom. Anim. Toxins incl. Trop. Dis 12, 1943. Cunningham, J.G., 2004. Tratado de Fisiologia Veterinaria, third ed. Guanabara Koogan, Rio de Janeiro. Cupo, P., Azevedo-Marques, M.M., Hering, S.E., 2003. Escorpionismo. In: Cardoso, J.L.C., Frana, F.O.S., Wen, F.H., Malaque, C.M.S., Haddad Jr., V. (Eds.), Animais Peonhentos no Brasil: Biologia Clnica e Terapeutica dos Acidentes. Salvier, Sao Paulo, pp. 198208. Cupo, P., Figueiredo, A.B., Pintya Filho, A., Pintya, A.O., Tavares Jr., C.A., Caligaris, F., Marin-Neto, J.A., Hering, S.E., Simoes, M.V., 2007. Acute left ventricular dysfunction of severe scorpion envenomation is related to myocardial perfusion disturbance. Int. J. Cardiol. 116, 98106. Cupo, P., Hering, S.E., 2002. Cardiac troponin I release after severe scorpion envenoming by Tityus serrulatus. Toxicon 40, 823830. Cupo, P., Jurca, M., Azevedo-Marques, M.M., Oliveira, J.S.M., Hering, S.E., 1994. Severe scorpion envenomation in Brazil. Clinical, laboratorial and anatomopathological aspects. Inst. Med. Trop. Sao Paulo 36, 6776. Diniz, P.P.V.P., Schwartz, D.S., Collicchio-Zuanaze, R.C., 2007. Cardiac trauma conrmed by cardiac markers in dog: two cases reports. Arq. Bras. Med. Vet. Zoot. 59, 8589. El-Asmar, M.F., 1984. Metabolic effect of scorpion venom. In: Tu, A.T. (Ed.), Handbook of Natural Toxins, Insects, Poisons, Allergens and other Invertebrate Venoms, vol. 2. Marcel Dekker, New York, pp. 551575. Finco, D.R., 1997. Kidney function. In: Kaneko, J.J., Harvey, J.W., Bruss, M.L. (Eds.), Clinical Biochemistry of Domestic Animals, fth ed. Academic Press, San Diego, pp. 441484. Fletcher, P.L., Fletcher, M., Fainter, L.K., Terrian, D.M., 1996. Action of new world scorpion venom and its neurotoxins in secretion. Toxicon 34, 13991411. Fukuhara, Y.D.M., Dellalibera-Joviliano, R., Cunha, F.Q.C., Reis, M.L., Donadi, E.A., 2004. The kinin system in the envenomation caused by the Tityus serrulatus scorpion sting. Toxicol. Appl. Pharmacol 196, 390395. Fukuhara, Y.D.M., Reis, M.L., Dellalibera-Joviliano, R., Cunha, F.Q.C., Donadie, E.A., 2003. Increased plasma levels of IL-1b, IL-6, IL-8, IL-10, and TNF-a in patients moderately or severely envenomed by Tityus serrulatus scorpion sting. Toxicon 41, 4955. Gazarian, K.G., Gazarian, T., Hernandez, R., Possani, L.D., 2005. Immunology of scorpion toxins and perspectives for generation of antivenom vaccines. Vaccine 23, 33573368. Hassan, F., 1984. Production of scorpion anivenin. In: Tu, A.T. (Ed.), Handbook of Natural Toxins, Insects, Poisons, Allergens and other Invertebrate Venoms, vol. 2. Marcel Dekker, New York, pp. 577605. Hetland, ., Dickstein, K., 1998. Cardiac troponins I and T in patients with suspected acute coronary syndrome: a comparative study in a routine setting. Clin. Chem. 44, 14301436. Ismail, M., 1995. The scorpion envenoming syndrome. Toxicon 33, 825858. Ismail, M., Abd-Elsalam, M.A., 1988. Are the toxicological effects of scorpion envenomation related to tissue venom concentration? Toxicon 26, 233256. Jain, N.C., 1993. Essentials of Veterinary Hematology. Lea and Febiger, Philadelphia. Kaneko, J.J., 1997. Carboydrate metabolism and its diseases. In: Kaneko, J.J., Harvey, J.W., Bruss, M.L. (Eds.), Clinical Biochemistry of Domestic Animals, fth ed. Academic Press, San Diego, pp. 4581. Kaneko, J.J., Hervey, J.W., Bruss, M.L., 1997. Appendixes. In: Kaneko, J.J., Harvey, J.W., Bruss, M.L. (Eds.), Clinical Biochemistry of Domestic Animals, fth ed. Academic Press, San Diego, pp. 885905. Kramer, J.W., Hoffmann, W.E., 1997. Clinical enzymology. In: Kaneko, J.J., Harvey, J.W., Bruss, M.L. (Eds.), Clinical Biochemistry of Domestic Animals, fth ed. Academic Press, San Diego, pp. 303325. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265275. Magalhaes, M.M., Garbacio, V.L., Almeida, M.B., Braz, A.C.A., MoraesSantos, T., Freire-Maia, L., Cunha-Melo, J.R., 2000. Acid-base balance following Tityus serrulatus scorpion envenoming in anaesthetized rats. Toxicon 38, 855864. Magalhaes, M.M., Pereira, M.E.S., Amaral, C.F.S., Rezende, N.A., Campolina, D., Bucaretchi, F., Gazzinelli, R.T., Cunha-Melo, J.R., 1999. Serum levels of cytokines in patients envenomed by Tityus serrulatus scorpion sting. Toxicon 37, 11551164. Meki, A.R.A.M., Mohamed, Z.M.M., El-Deen, H.M.M., 2003. Signicance of assessment of serum cardiac troponin I and interleukin-8 in scorpion envenomed children. Toxicon 41, 129137.

Considering that the half-lives of insulin and cortisol are short (510 and 60 min, respectively) (Kaneko, 1997; Cunningham, 2004), and that the response to stress and hyperglycaemia is rapid, the secretion and removal of these two hormones occurred within a short time interval. There were, therefore, large oscillations in the concentrations of these two hormones during the period immediately following inoculation of venom (up to 6 h), at exactly the time when the animals expressed more pain and when glycaemia was at the highest level. The results presented here indicate that energy metabolism was not impaired by the inoculation of venom, since acid-base balance was normal and no myocardial lesions were detected. Although catecholamine discharge had no effect on insulin secretion, this discharge could have inuence in hyperglycaemia by increasing the hepatic mobilisation of glycogen and muscle protein catabolism. It is important to note that the high dose of venom used may have maximized the painful reaction, and consequently the discharge of cortisol and catecholamines, which, however, was not able to inhibit pancreatic insulin secretion. We conclude that 250 mg/kg of venom of Tityus serrulatus causes acute and discrete changes in dog biochemical prole, which is primarily related to the stress of pain without evidence of electrolyte imbalances, kidney and myocardial muscle damage as normally described in humans. Acknowledgments The authors wish to thank the Coordenaao de Aper feioamento de Pessoal de Nvel Superior (CAPES) for a scholarship awarded to E.L. Ribeiro, and the Fundaao de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Cientco e Tecnologico (CNPq) for nancial support for this project. Conict of interest There are no conicts of interest in the content of this work. References
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