Effect of scorpion Leiurus quinquestriatus (H&E) venom on rat's heart rate and blood pressure

Toxicology Letters, 61 (1992) lllll21 0 1992 Elsevier Science Publishers B.V. All rights reserved 0378-4274/92/$ 5.00

111

TOXLET 02721

Effect of scorpion Leiurus quinquestriatus (H&E) venom on rat’s heart rate and blood pressure

Mohamed A.A. Omran, Mohamed S. Abdel-Rahman

and Z.I. Nabil

Pharmacology and Toxicology Department, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ (USA)

(Received 1 November 1991) (Accepted 22 January 1992) Key words: Scorpion venom; Heart rate; Blood pressure; Propranolol; Atropine SUMMARY The effects of scorpion Leiurus quinquestriarus (H&E) venom collected from the South Sinai region, Egypt, on heart rate (HR) and mean arterial pressure (MAP) were studied after intramuscular administration of 3 different doses (100, 200 and 400 ,@kg) to anesthetized rats. The effects of adrenergic and cholinergic blocking agents on the venom-induced HR and MAP changes were also evaluated. In two groups of rats, propranolol or atropine were given before the venom administration. In the third group the venom was given before the injection of propranolol and atropine in combination. HR was measured by using a cardiotachometer coupler connected to an ECG coupler. MAP was calculated from the recorded arterial blood pressure (ABP) after catheterization of the left common carotid artery. Venom doses of 100 and 200 pg/kg produced tachycardia with a dose-response relationship, whereas 400 pg!kg evoked sinus tachycardia followed by bradycardia then tachycardia. MAP was elevated after the administration of each dose and reached its maximum value after 60 min with a dose-response relationship. Sinus, atria1 and ventricular arrhythmias were observed from the recorded ECG during the time studied. This study revealed that the venom has pressor and depressor effects which are mediated through the autonomic nervous system. Propranolol reduced the stimulatory effects of the highest dose of the venom while atropine was effective in eliminating the depressor effect of the venom on HR. The arrhythmias induced by the venom were blocked by the injection of the two blockers and are assumed to be due to the release of catecholamines and acetylcholine.

INTRODUCTION

Despite the great number of scorpion species, only a few belonging to the Buthidae

Correspondence to: Dr. M.S. Abdel-Rahman, Department of Pharmacology and Toxicology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2714, USA.

112

family are considered very dangerous. The scorpion Leiurus quinquestriutus is among the most common and dangerous species in many parts of the world. An averagesized human adult would have less than a 50% chance of surviving on injection of 17 mg of the venom of the buthid Leiurus quinquestrktus [I]. This scorpion is also found in the South Sinai region. Egypt, which has a relatively dense population and poisoning due to this species is a considerable problem in this area. The actual effects of the sting depend upon a number of factors such as immunological disposition of the victim, thickness of the skin, weight of the victim, circulation through the affected part of the body, and also the amount of venom available in the scorpion at the time of stinging [2]. Different species of scorpions have different kinds of poisons and these differ in chemical structure and physiological effects. In fact, scorpions of the same species from different geographical areas have venoms that vary in their chemical composition and their toxic effects [ 1,3]. Scorpion venoms contain mainly neurotoxins, as well as cardiotoxins [4]. Anderson [l] has reported that the toxic materials of the venom are protein in nature. The number of different kinds of proteins known from scorpion venoms ranges from 5 to 16, and their toxicity depends on the nature of the toxic protein present. The venom from the most dangerous species contains at least 5 toxic proteins. Each toxic protein consists of a single polypeptide chain cross-linked by 4 disulfide bridges. This chain contains from 56 to 69 amino acids. The effect of the scorpion venom is not necessarily due to the direct effect of its components. The venom may manifest its action (autopharmacological action) by releasing some tissue components [4]. It may act by interfering with the nervous system including the autonomic as well as the neuromuscular systems [5]. It may also affect the cardiovascular system [6], the respiratory system, and the homeostatic mechanism of the body through influencing hormonal release and/or acting on the higher centers [7]. The distribution of Leiurus quinquestriutus venom in the rat tissue after intramuscular or intraperitoneal administration was found to be similar [8]. The relatively high concentration of the venom in the lungs and heart may indicate that these organs are among the primary sites for the action of the venom. An investigation by Gueron and Yarom [9] suggested that the scorpion venom may have a direct myocardial effect in addition to its stimulatory effects on the adrenals and autonomic nervous system. Experimental data and clinical investigations by Freire-Maia and Campos [lo] have shown that scorpion poisoning is a very complex syndrome. Depending on the dose of venom injected, sinus tachycardia and arterial hypertension could be followed by atrioventricular block and arterial hypotension. On the other hand, the rhythmic disturbances do not occur alone, but have a simultaneous effect on the systemic arterial pressure. Treatment of a severe scorpion sting consists mainly of a specific antivenom [lo]. Administration of anticholinergic drugs or CI- or /?-adrenergic blocking agents depends on the specific symptoms and signs [l I]. Sofer and Gueron [12] have stated

113

that experimental and human studies suggest that a-adrenergic and /I-adrenergic blocking agents should be used for the treatment of hypertension and related cardiovascular events induced by scorpion envenomization. In the mean time, some investigators [13] have concluded that the relative role of /?-adrenergic receptor activation due to venom administration is unknown. This study was conducted to investigate the effects of different doses of scorpion Leiurus quinquestriutus venom on heart rate and blood pressure as well as the mechanism of this effect by using sympathetic and parasympathetic blocking agents. MATERIALS

AND METHODS

Animals Male Sprague-Dawley rats (350400 g) were purchased from Taconic Farms (Germantown, NY) and housed 3 per cage at 22°C and humidity of 50% with a 12-h light/dark cycle. Purina laboratory chow and tap water were provided ad libitum. Scorpion venom Scorpion Leiurus quinquestriutus (H&E) was collected from the Sinai area (Egypt). The venom was obtained by electrical stimulation of the telson, suspended in distilled water and the supernatant was lyophilized. The lyophilized venom was kept in a desiccator over calcium chloride, and when required it was reconstituted by the addition of saline (0.9% NaCl). Effect of the venom Animals were divided into 4 groups (5 animals per group). Three groups were injected intramuscularly with the scorpion venom (100, 200 and 400 pug/kg), respectively. Intramuscular administration most probably reproduces the natural effects of the sting [7,14,15]. The fourth group was treated with the vehicle (saline) and the study proceeded under the same conditions. Effect of sympathetic and parasympathetic blocking agents Two groups of rats were treated intraperitoneally with propranolol or atropine (1 mg/kg) 20 min prior to venom injection (400 puglkg, i.m.). Another group was administered propranolol and atropine in combination (1 mg/kg each i.p.) 30 min after venom injection (400 @kg, i.m.). ECG, heart rate and blood pressure measurements Rats were anesthetized with urethane (1.25 g/kg, i.p.) obtained from Sigma Chemical Company (St. Louis, MO). ECG was taken according to the method described by Buchman et al. [16] using bipolar electrodes, limb lead II, in the direction of the anatomical heart axis. The ECG electrodes were connected to an ECG coupler (Beckman Inc. Type 9855) for recording the ECG. Heart rate was recorded by connecting the output from the ECG coupler to a cardiotachometer coupler (Beckman, Inc. Type 9857B). According to the method described by Popovic and Popovic [17], PE-50 catheter tubing (Clay Adams, Parsippany, NJ) was introduced into the left common carotid artery in the same animal. The carotid catheter was connected to a

114

Statham P23AC pressure transducer (Gould-Statham Instruments, Inc., Hato Rey, Puerto Rico) for blood pressure recording and then attached to a strain gauge coupler (Beckman Inc. Type 9803). All the above couplers were attached to a Beckman R511A 4-channel recorder (Beckman Instruments, Inc., Somerset, NJ). After the surgical preparation, the rats were left for 30 min before recording the data. Heart rate and blood pressure [systolic (sBP) and diastolic (dBP)] were recorded for 10 min to serve as controls prior to treatment. Readings were taken for 4-h sessions. Mean arterial pressure (MAP) was calculated by the following equation: MAP = (sBP - dBP)/3 + dBP. Data analysis Differences between the mean values of the groups were compared by one-way analysis of variance (ANOVA) with Scheffe’s test. In all analyses the level of significance was chosen as P < 0.05. RESULTS

The data obtained from the control group showed that there were no significant differences between the values measured before and after treatment for the parameters tested. This indicates that the stress and the surgical operation had no effect on the animals during the entire time course. Effect of the venom Table I shows the effect of the scorpion venom on the heart rate in rats. There is a gradual increase in heart rate as a result of injection of 100 and 200 ,uglkg doses. This

TABLE EFFECT

I OF SCORPION

Time (h)

VENOM

ON THE HEART

Control

RATE

Dose @g/kg) 100

0.00 0.25 0.50 1.00 1.50 2.00

325 k 8.0” 328 i 10.0

318 f 5.0 333 * 5.0

328 f 11.0 332 5 9.0

336 f 5.0 345 t 8.0

325 k 10.0

350 f 7.0 362 k 9.0

329 + 9.0 324 k 12.0 321 F 11.0

3.00 4.00

200

400

320 + 19.0

328 + 21.0

361 f 10.0 369 + 17.0 375 * 9.0’

363 k 20.0 372 f 20.0 321 k 12.0

385 + 20.0’

263 k 11.0’ 278 f 13.0’

364 k 10.0

387 k 11.0’ 416 f 12.0’

376 i 9.0’

422 zk 13.0’

“Values are the mean k SE of the heart rate (beat/min.) from 5 animals intramuscular administration of lyophilized Leiurus quinquestriatusvenom. *Significantly

different

from control

group,

ANOVA,

P c: 0.05.

342 + 17.0 398 f 13.0’ per group

before

and after

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increase became significant 4 h after the administration of the 100 ,uuglkgdose. On the other hand, at the 200 pg/kg dose, the heart rate was significantly increased as early as 1 h and was sustained until the end of the time course. It is worth noting that injection of the highest dose (400 pg/kg) produced a 3-phase effect, namely initial tachycardia followed by a significant decrease in heart rate and then sustained tachycardia which became significant by the end of the time course. The effect of venom on the MAP is shown in Table II. The data from this experiment indicate that there is a dose-response relationship. The maximum elevation in MAP was reached 1 h after treatment in all groups. At the lowest dose of venom (100 &kg) the change in MAP became significant 1 and 1.5 h after injection compared to the control group. However, administration of the two higher doses (200 yglkg or 400 yglkg) resulted in a significant elevation as early as 0.5 and 0.25 h, respectively, from the time of administration. This significant increase continued during the study period. Several cases of cardiac disorders were noticed as a result of treatment with the 200 and 400 puglkg doses. Some of these cases are illustrated in Figure 1. Table III lists the type and number of these cases. Each case was considered to be tabulated as one incident for each animal during the whole time course, even if it was notied several times in some EGG traces. Effect of the sympathetic andparasympathetic blocking agents Figure 2 shows the effect of atropine or propranolol pretreatment on the venominduced changes in heart rate. It is clear that heart rate increased gradually during the entire time course in the case of atropine administration. This increase became significantly different from the venom-injected group alone (400 lug/kg) from 1 to 4 h.

TABLE II EFFECT OF SCORPION VENOM ON THE MEAN ARTERIAL BLOOD PRESSURE Time (h)

Control

Dose @g/kg) 100

0.00

0.25 0.50 1.oo 1.50 2.00 3.00 4.00

II f 75 f 14 f 16 k 15 f 15 + II f 78 +

3.3 3.3 3.2 2.8 3.2 3.4 3.1 4.0

15 f 76 It 96 k 105 + 105 f 93 f 82 k 80 +

200 9.1 10.4 6.1 5.0’ 4.6’ 1.5 8.2 6.0

84 f 104 + 123 f 131 + 120 + 121 + 113 f 105 f

400 5.2 5.0 6.8’ 6.0’ 3.9’ 4.1’ 6.8’ 8.5’

94 + 115 f 135 f 152 + 139 f 133 + 132 f 127 +

6.7 11.1’ 11.8’ 6.0’ 9.7’ 5.5’ 5.3’ 4.5’

a Values are the mean f SE of the mean arterial blood pressure (mmHg) from 5 animals per group before and after intramuscular administration of the lyophilized Leiurus quinquestriutus venom. *Significantly different from control group, ANOVA, P < 0.05.

116

Fig. 1.Examples of the cardiac disorders recorded as a result of intramuscular (200 and 400 ,@kg). A = Sinus arrhythmia (arrow); B = AV nodal rhythm (arrow);

D = premature

ventricular

contraction

abnormality

(arrow).

tracing;

injection of scorpion venom (asterisk); C = sinus arrest

Paper speed = 50 mm/s.

3 = recovery

I = control tracing; 2 =

tracing.

Atropine injection prevents the appearance of the second phase (bradycardia) which was recorded after injection of the venom alone. On the other hand, propranolol pretreatment caused a decrease in the heart rate even after injection of the venom. At the same time the early and late tachycardia (Phases I and III) were prevented due to propranolol administration. The only significant differences between this group and that injected with venom alone were observed 0.25, 0.5 and 4 h after venom administration. Figure 3 shows the effect on the heart rate of administration of propranolol and atropine in combination 30 min after venom injection. Treatment with both drugs significantly prevented the dramatic effect of the venom on the heart rate and the second and third phase effects were not recorded at the same time points. The heart rate almost returned to the initial values (O-time) by the end of the time course.

117

The changes in MAP in the group injected with venom alone and the propranololpretreated group (20 min before venom administration) are shown in Figure 4. Propranolol injection normalized MAP and returned it to initial values 3 h after venom administration. Also the highest level of MAP which was noted after 1 h in the case of venom injection alone was reduced significantly by propranolol pretreatment in the other group. At the same time, a significant decrease in MAP was observed 3 and 4 h after venom injection when compared with the elevated values in the venom-administered group at the same time points. It is important to emphasize that treatment with sympathetic and parasympathetic blocking agents attenuated the appearance of the cardiac disorders which were recorded after injection of the venom alone. DISCUSSION

In several parts of the world, such as the Southwestern United States, South America, Mexico, India, North Africa and the Middle East, scorpion venom still represents a medical hazard to the population living in those areas. Among the dangerous species found in those areas are Centruroides sculpturatus, Buthus tamulus, Tityus serrulatus, Buthus occitanus, Androctonus crassicauda and Leiurus quinquestriatus. TABLE III INCIDENCE OF DIFFERENT CASES OF CARDIAC DISORDERS NOTED AS A RESULT OF INTRAMUSCULAR INJECTION OF SCORPION VENOM Type of disorder

Dose @g/kg) 200

400

Sinus arrhythmias Sinus tachycardia Sinus bradycardia Sinus arrhythmia Sinus arrest Wandering pace maker

5” 1 _ 2 2

5 5 4 4 4

Atria1 arrhythmias Atrial fibrillation

3

3

AV nodal rhythm

2

4

Ventricular arrhythmias Premature ventricular contraction (PVC)

2

4

a Number of cases observed in the ECG tracing from 5 animals per treatment group.

118

-v-@ooLg&?l before venom . betore Venom

-i-Atmp.

+Pmp.

250

150 -1

1

0

2

TIME Fig. 2. Effect injection. scorpion

Prop.

on heart

rate of propranolol

or Atrop.

(1 mg/kg,

(Prop.)

or atropine

-

300

-

200 ' -1

I 0

(Atrop.)

administration

(i.p.) 20 min. before administration

before

venom

of 400 ,ug/kg

mean + SE of 5 animals per group. *Statistical significant versus venom administration only; ANOVA, P < 0.05.

5ooi 400

i

4

(hours)

each) were injected

venom (i.m.). Symbols represent ferences from the same time-points

3

dif-

-e--v-(~LuJ/Ql

i

I 1

---o_venom

I 2

TIME

I 3

before

rF%op./Atmp.).

I 4

5

(hours)

Fig. 3. Effect of propranolol (Prop.)/atropine (Atrop.) administration in combination after venom injection on heart rate. Prop./Atrop. (1 mg/kg, each) were injected (i.p.) 30 min after administration of 400 /@kg scorpion venom (i.m.). Symbols represent mean i- SE of 5 animals per group. *Statistical significant differences from the same time-points versus venom administration only; ANOVA, P < 0.05.

119

-1

0

I

2

TIME

3

4

5

(hours}

Fig. 4. Effect of propranolol (Prop.) administration before venom injection on mean arterial pressure. Prop. (1 mgikg) was injected (i.p.) 20 min before administration of 400 fig/kg scorpion venom (i.m.). Symbols represent mean t SE of 5 animals per group. *Statistical significant differences from the same time-points versus venom administration only; ANOVA, P * 0.05.

In the present study, scorpion venom increased the heart rate and evoked a slight and severe tachycardia at 100 and 200 &kg doses, respectively, while the highest dose (400 ygikg) produced a 3-phase effect: tachycardia (Phase I> followed by a short-lasting bradycardia (Phase II) and a final sustained tachycardia (Phase III}. Our data on the lower doses are in agreement with the study of Freire-Maia et al. [18]. They reported that the injection of purified scorpion Tityus serrulatus toxin in anesthetized rats produced sinus tachycardia at 5&200 ,ug/kg doses. However, the same authors reported that higher doses (400 and 800 ,ug/kg) caused a sinus bradycardia only, which is different from our results. On the other hand, the report of Zlotkin et al. 1191is in agreement with our report using the high dose. they demonstrated that the initial cardiac effect is stimulatory followed by an inhibitory effect in the case of injection of high doses of venom. The sinus tachycardia may be caused by activation of /?-adrenergic receptors in the heart and the bradycardia could be due to release of acetylcholine by the action of the toxin on vagal ganglia and postganglionic nerve endings in the heart. Also, Ismail et al. [20] have reported that the effect of ~~t~~~ occitanus venom on isolated hearts appears to be mediated mainly through stimulation of the autonomic nervous system with predominance of sympathetic stimulation and release of catecholamines. It is worth noting that this effect was observed in humans as well. Cueron and Ovsyshcher [I 1] reported an initial sinus tachycardia in patients following a scorpion sting, although sinus bradycardia was also recorded. The administration of atropine 20 min before venom (400 @kg) injection blocked

I 20

the second negative chronotropic effect (Phase II) and potentiated the positive chronotropic effect. This effect indicates that the bradycardia caused by the venom might be due to stimulation of the vagal ganglia and postganglionic nerve endings of the heart [7,20,21]. On the other hand, the tachycardia (Phases I and 111) is due to sympathetic stimulation and release of tissue and medullary chatecholamines because propranolol administration prior to the venom (400,~glkg) injection produced bradycardia only. Treatment of rats with propranolol and atropine in combination after venom administration reduced to some extent the adrenergic and cholinergic effects of the venom. Ismail and Abd-Elsalam [22] concluded that treatment of rabbits with both atropine and propranolol protected the animals from the lethal action of the scorpion venom and prevented the cardiovascular effect. Hypertension is considered to be a characteristic effect of all the Buthid family venoms in a wide variety of test animals [ 18,20,23,24]. Our data have shown that there is a doseeresponse effect related to the hypertension which may be due to the release of catecholamines due to the effect of the venom [25]. Zlotkin et al. [19] have reported that catecholamines may elevate the blood pressure either by vasoconstriction or increasing the heart rate. The hypertensive effect of the venom was potentiated in rats pretreated with atropine. This could be due to increasing the cardiac output and/or excessive stimulation of the P-receptors in the myocardium as a result of vagal-effect blockade, thus leaving the P-receptor effect unopposed. This phenomenon is supported by the data obtained from our study after pretreatment with propranolol which almost reversed the hypertension and normalized it again by the end of the time course. However, the use of propranolol and atropine together after venom administration did not completely abolish the elevated MAP. This may be due to the time of blocker injection and at the same time the effect of the venom on the unblocked a-receptors and/or by increasing the circulating adrenal catecholamines. In conclusion, this study demonstrates that the venom of the South Sinai scorpion, Leiurus quinquestriutus (H&E), activates both sympathetic and parasympathetic fibers and that the initial stimulatory cardiac effect is followed by an inhibitory effect (in case of the high dose) as suggested by Zlotkin et al. [ 191 and not in the reverse order as shown by Grupp et al. [26]. Our data indicated that the changes which were observed in the heart rate were related to the adrenergic and cholinergic effects of the venom. At the same time, propranolol decreased the magnitude of the pressor effect whereas atropine antagonizes the depressor effects of the venom.

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