Effect of Androctonus bicolor scorpion venom on serum electrolytes in rats: A 24-h time–course study

Abstract

Black fat-tailed scorpion (Androctonus bicolor) belongs to the family Buthidae and is one of the most venomous scorpions in the world. The effects of A. bicolor venom on serum electrolytes were not known and therefore investigated in this study. Adult male Wistar rats were randomly divided into seven groups with five animals in each group. One of the groups served as control and received vehicle only. The animals in the remaining groups received a single subcutaneous injection of crude A. bicolor venom (200 μg/kg bodyweight) and were killed at different time intervals including 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h after venom injection. The results showed that scorpion venom caused significant increase in serum sodium levels within 30 min after injection which slightly subsided after 1 h and then persisted over 24 h. Serum potassium levels continued to significantly increase until 4 h and then slightly subsided. There were significant decreases in serum magnesium (Mg⁺) levels following scorpion venom injection, at all the time points during the course of study. Serum calcium levels were significantly increased during the entire course of study, whereas serum chloride was significantly decreased. In conclusion, A. bicolor envenomation in rats caused severe and persistent hypomagnesemia with accompanied hypernatremia, hyperkalemia, and hypercalcemia. It is important to measure serum Mg⁺ levels in victims of scorpion envenomation, and patients with severe Mg⁺ deficiency should be treated accordingly.

Introduction

Black fat-tailed scorpion (Androctonus bicolor) belongs to the family Buthidae and is one of the most venomous scorpions in the world. These scorpions typically possess black and brown coloration, tend to move very fast, and have an aggressive nature. Both black and brown scorpions of this species are dangerous and may cause multisystem manifestations, especially in young children. ¹ Recently, a novel type of venom peptide with six disulfide bridges, referred to as Androcin, was identified from the scorpion A. bicolor using a complementary DNA library strategy. ² Toxicological analysis using the recombinant Androcin peptide revealed that Androcin is able to induce severe akinesia and anxiety-like symptoms in mice, thereby providing the scorpion with another tool to subdue animals. ² Cusinato et al ³ evaluated the hematological changes induced by Tityus serrulatus scorpion venom, and their findings demonstrated that this venom is able to induce severe hematological changes that appear within the first hours after envenoming, justifying a prompt medical attention to avoid worsening of clinical symptoms. However, there is no report on electrolyte imbalance resulting from A. bicolor envenomation in human or experimental animals. We therefore conducted a time–course study to investigate the alterations in serum electrolytes following a single subcutaneous injection of A. bicolor venom in rats.

Materials and methods

A. bicolor scorpions were collected from the Riyadh region. They were kept in large plastic boxes at room temperature and fed with mealworms and received water daily. The scorpions were milked by electrical stimulation and the venom was stored at −80°C.

Adult male Wistar rats (180–220 g bodyweight) grown in our animal house facility were used. The animals were placed in polycarbonate cages, kept in a room maintained at 23 ± 1°C with 12-h light/12-h dark cycles and free access to standard laboratory food and tap water. The animals were randomly divided into seven groups with five animals in each group. Group 0 served as control and received vehicle only. All the animals in the remaining groups received a single subcutaneous injection of crude venom (200 μg/kg bodyweight) and killed at different time intervals as follows: 30 min (group 1), 1 h (group 2), 2 h (group 3), 4 h (group 4), 8 h (group 5), and 24 h (group 6) after venom injection. Blood samples were collected by cardiac puncture, sera separated, and stored at 4°C until analyzed. The experimental protocol was approved by the Institutional Research and Ethics Committee.

Serum electrolytes including sodium (Na⁺), potassium (K⁺), magnesium (Mg²⁺), calcium (Ca²⁺), and chloride (Cl^–) were determined using commercial kits purchased from United Diagnostic Industry (Dammam, Saudi Arabia). We used disposable cuvettes and an ultraviolet (UV)–visible spectrophotometer (Shimadzu UV-160A, Japan) for absorbance measurements according to the manufacturer’s instructions.

The data were analyzed by analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test using the Statistical Package for Social Sciences software (SPSS Inc., Chicago, Illinois, USA). Pearson’s test was used for correlation studies. The values of p < 0.05 were considered as statistically significant.

Results

A single subcutaneous injection of A. bicolor venom (200 μg/kg) in rats caused significant increase in serum Na⁺ levels at 30 min after injection, and this increase slightly subsided after 1 h and then persisted over 24 h (ANOVA F = 10.65, p < 0.001; Table 1, Figure 1). There was a continued significant increase in serum K⁺ levels until 4 h, followed by a slight subsidence, though significant hyperkalemia persisted over 24 h (ANOVA F = 17.54, p < 0.001). There were significant decreases in serum Mg²⁺ levels following scorpion venom injection, at all the time points during the course of study (ANOVA F = 40.79, p < 0.001). Serum Ca²⁺ levels were significantly increased during the entire course of the study (ANOVA F = 4.15, p < 0.01), whereas serum Cl^– level was significantly decreased (ANOVA F = 5.92, p < 0.001; Table 1). The percent time–course changes in serum electrolytes following scorpion venom envenomation are shown in Figure 1. The maximum post-dosing increases in Na⁺, K⁺ and Ca²⁺ levels were 46.0% (30 min), 74.1% (4 h), and 30.5% (1 h), respectively. The maximum decreases in serum Mg²⁺ and Cl^– levels after venom injection were 50.3% (24 h) and 18.4% (4 h), respectively (Figure 1). There were significant inverse correlations of serum Mg²⁺ with Na⁺ (R = −0.677, p < 0.001), K⁺ (R = −0.603, p < 0.001), and Ca²⁺ (R = −0.432, p < 0.01), but significantly direct correlation with Cl^– levels (R = 0.424, p < 0.05).

Table 1.

Time–course effects of Androctonus bicolor scorpion venom on the levels of serum electrolytes in rats.^a

Groups	Na+ (mmol/l)	K+ (mmol/l)	Mg2+ (mg/dl)	Ca2+ (mg/dl)	Cl− (mequiv./l)
G0 (control)	135.2 ± 1.46	5.10 ± 0.24	3.62 ± 0.26	10.30 ± 0.96	103.0 ± 0.84
G1 (0.5 h)	197.4 ± 1.17b	7.06 ± 0.10b	1.86 ± 0.02b	12.72 ± 0.17c	95.0 ± 1.05
G2 (1 h)	174.8 ± 5.15b	7.70 ± 0.16b	1.84 ± 0.02b	13.44 ± 0.22d	86.6 ± 2.68d
G3 (2 h)	166.8 ± 8.07d	8.32 ± 0.40b	1.82 ± 0.02b	12.82 ± 0.14c	92.2 ± 4.77c
G4 (4 h)	179.2 ± 7.08b	8.88 ± 0.46b	2.24 ± 0.07b	11.16 ± 0.24c	84.0 ± 2.47d
G5 (8 h)	176.4 ± 8.20b	7.76 ± 0.21b	1.86 ± 0.04b	12.36 ± 1.12c	88.2 ± 2.01c
G6 (24 h)	181.6 ± 5.24b	7.52 ± 0.22b	1.80 ± 0.05b	13.36 ± 0.34d	96.6 ± 3.01

Na⁺: sodium ion; K⁺: potassium ion; Mg²⁺: magnesium ion; Ca²⁺: calcium ion; Cl⁻: chloride ion.

^aData are presented as mean ± SEM.

^bp < 0.001: versus control group (G0) using Dunnett’s test.

^cp < 0.05: versus control group (G0) using Dunnett’s test.

^dp < 0.01: versus control group (G0) using Dunnett’s test.

Figure 1.

Percent change in serum electrolyte levels at different times post-dosing of scorpion venom. Baseline (control) values were set as 100. ^ap < 0.05; ^bp < 0.01; ^cp < 0.001: versus control group using Dunnett’s test.

Discussion

The findings of this study showed peculiar time–course pattern of serum electrolytes in rats injected with a single dose of A. bicolor venom. There were increasing trends in serum Na⁺, K⁺ and Ca²⁺ levels while decreasing trends in serum Mg²⁺ and Cl^– levels (Table 1). When compared with the previously reported data on changes in serum electrolytes following envenomation with different scorpion venoms, the effects of A. bicolor venom are quite unique and interesting. T. serrulatus scorpion toxin injection resulted in hyperosmolality, hyperkalemia, and hypermagnesemia in rats. ⁴ Cusinato et al. ³ did not observe any significant changes in serum Na⁺ and Ca²⁺ levels, but an increase in Mg²⁺ and K⁺ levels following a single intraperitoneal injection of T. serrulatus venom (500 µg/kg) in rats. Omran and Abdel-Rahman ⁵ have noticed significant decrease in serum Ca²⁺ and K⁺ levels, 4 h after the administration of 100–400 µg/kg doses of Leiurus quinquestriatus scorpion venom in rats. Same doses of Palamneus gravimanus venom also significantly reduced serum Ca²⁺ and K⁺ levels, 4 h after the intramuscular injection of scorpion venom in rats. ⁶ Administration of Buthus tamulus scorpion venom in the doses of 2 and 4 mg/kg body weight did not produce any change in serum electrolytes of dogs. ⁷

The most striking observation of this study was a severe and persistent hypomagnesemia accompanied with severe hyperkalemia in rats injected with A. bicolor venom. Mg is the fourth most abundant cation in the body and the second most abundant intracellular cation after K. Mg is important for the proper functioning of various metabolic pathways and ion channels, so disturbances in Mg concentration can cause clinical problems. Under normal conditions, the body maintains constant circulating concentrations of Mg in the blood. Mg homeostasis depends on the balance between its intestinal absorption and renal excretion, with kidney tubules playing important role in control mechanism. ⁸ Mg is involved in over 300 enzymatic reactions. It is needed in energy metabolism, glucose utilization, protein synthesis, fatty acid synthesis and breakdown, muscle contraction, all adenosine triphosphatase (ATPase) functions, for almost all hormonal reactions, and in the maintenance of cellular ionic balance. ⁹ Because Mg is required for the proper functioning of the Na⁺/K⁺ ATPase pump, its deficiency causes an increase in intracellular Na⁺ and allows K⁺ ions to leak out of cells. ⁹ Loss of intracellular K also occurs in the renal tubules. ¹⁰ This can lead to a hypokalemia that fairly responds to Mg replacement therapy. ⁹ However, in our study, serum K⁺ levels were significantly increased by A. bicolor envenomation in rats (Figure 1).

We observed a highly significant inverse correlation between serum Mg²⁺ and Ca²⁺ levels. Mg is known to affect Ca homeostasis, as many Ca²⁺ channels are dependent on Mg. Higher concentrations of the intracellular Mg inhibit Ca transport into the cell, whereas Mg deficiency results in an increase of the intracellular Ca²⁺ levels. Mg is also required for the release and proper action of parathyroid hormone. ⁹ Patients with hypomagnesemia may also have low plasma Ca²⁺ levels that remain refractory to Ca supplementation until the Mg deficiency is replenished. ⁹ However, this was not the case in our study, as we observed severe hypomagnesemia accompanied with mild-to- moderate hypercalcemia in rats injected with A. bicolor venom (Table 1). The usual role of Mg²⁺ in the Na⁺-K⁺ ATPase pump and Ca²⁺-blocking activity is impaired by hypomagnesemia, leading to membrane destabilization and hyperexcitability. ¹⁰ With severe hypomagnesemia, patients can have tetany and seizures. The effect on the myocardium is an increase in atrial and ventricular arrhythmias. Some ventricular arrhythmias caused by hypomagnesemia only respond to treatment with Mg. ¹¹ Administration of Mg has been found to be useful for the treatment of the arrhythmias, hypertension, and other disorders associated with Buthinae scorpion envenomation. ¹²

In conclusion, A. bicolor envenomation in rats caused severe and persistent hypomagnesemia with accompanied severe hyperkalemia and moderate hypernatremia and hypercalcemia. Disturbances in Mg²⁺ homeostasis can lead to serious conditions, some of which are only amenable to treatment with Mg. It is important to include serum Mg²⁺ in the routine serum electrolyte test (Na⁺, K⁺, Ca²⁺, and Cl^–) in victims of scorpion envenomation, and patients with severe Mg deficiency should be treated accordingly.