Age effects on the pharmacokinetics of tityustoxin from Tityus serrulatus scorpion venom in rats

Nunan, E.A.; Arya, V.; Hochhaus, G.; Cardoso, V.N.; Moraes-Santos, T.

doi:10.1590/S0100-879X2004000300016

Abstract

The pharmacokinetics of scorpion venom and its toxins has been investigated in experimental models using adult animals, although, severe scorpion accidents are associated more frequently with children. We compared the effect of age on the pharmacokinetics of tityustoxin, one of the most active principles of Tityus serrulatus venom, in young male/female rats (21-22 days old, N = 5-8) and in adult male rats (150-160 days old, N = 5-8). Tityustoxin (6 µg) labeled with 99mTechnetium was administered subcutaneously to young and adult rats. The plasma concentration vs time data were subjected to non-compartmental pharmacokinetic analysis to obtain estimates of various pharmacokinetic parameters such as total body clearance (CL/F), distribution volume (Vd/F), area under the curve (AUC), and mean residence time. The data were analyzed with and without considering body weight. The data without correction for body weight showed a higher Cmax (62.30 ± 7.07 vs 12.71 ± 2.11 ng/ml, P < 0.05) and AUC (296.49 ± 21.09 vs 55.96 ± 5.41 ng h-1 ml-1, P < 0.05) and lower Tmax (0.64 ± 0.19 vs 2.44 ± 0.49 h, P < 0.05) in young rats. Furthermore, Vd/F (0.15 vs 0.42 l/kg) and CL/F (0.02 ± 0.001 vs 0.11 ± 0.01 l h-1 kg-1, P < 0.05) were lower in young rats. However, when the data were reanalyzed taking body weight into consideration, the Cmax (40.43 ± 3.25 vs 78.21 ± 11.23 ng kg-1 ml-1, P < 0.05) and AUC (182.27 ± 11.74 vs 344.62 ± 32.11 ng h-1 ml-1, P < 0.05) were lower in young rats. The clearance (0.03 ± 0.002 vs 0.02 ± 0.002 l h-1 kg-1, P < 0.05) and Vd/F (0.210 vs 0.067 l/kg) were higher in young rats. The raw data (not adjusted for body weight) strongly suggest that age plays a pivotal role in the disposition of tityustoxin. Furthermore, our results also indicate that the differences in the severity of symptoms observed in children and adults after scorpion envenomation can be explained in part by differences in the pharmacokinetics of the toxin.

Scorpion venom; Tityustoxin; Pharmacokinetics; Age; Bootstrapping resampling; Non-compartmental analysis

Braz J Med Biol Res, March 2004, Volume 37(3) 385-390

Age effects on the pharmacokinetics of tityustoxin from Tityus serrulatus scorpion venom in rats

E.A. Nunan¹, V. Arya⁴, G. Hochhaus⁴, V.N. Cardoso²and T. Moraes-Santos³

¹Laboratório de Controle de Qualidade, Departamento de Produtos Farmacêuticos, ²Laboratório de Radioisótopos, Departamento de Análises Clínicas, ³Laboratório de Nutrição Experimental, Departamento de Alimentos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil

⁴Department of Pharmaceutics, College of Pharmacy, J.H. Millis Health Center, University of Florida, Gainesville, FL, USA

References

Correspondence and Footnotes ^{Correspondence and Footnotes} Correspondence and Footnotes

Abstract

The pharmacokinetics of scorpion venom and its toxins has been investigated in experimental models using adult animals, although, severe scorpion accidents are associated more frequently with children. We compared the effect of age on the pharmacokinetics of tityustoxin, one of the most active principles of Tityus serrulatus venom, in young male/female rats (21-22 days old, N = 5-8) and in adult male rats (150-160 days old, N = 5-8). Tityustoxin (6 µg) labeled with ^99mTechnetium was administered subcutaneously to young and adult rats. The plasma concentration vs time data were subjected to non-compartmental pharmacokinetic analysis to obtain estimates of various pharmacokinetic parameters such as total body clearance (CL/F), distribution volume (Vd/F), area under the curve (AUC), and mean residence time. The data were analyzed with and without considering body weight. The data without correction for body weight showed a higher C_max (62.30 ± 7.07 vs 12.71 ± 2.11 ng/ml, P < 0.05) and AUC (296.49 ± 21.09 vs 55.96 ± 5.41 ng h^-1 ml^-1, P < 0.05) and lower T_max (0.64 ± 0.19 vs 2.44 ± 0.49 h, P < 0.05) in young rats. Furthermore,

Vd/F (0.15 vs 0.42 l/kg) and CL/F (0.02 ± 0.001 vs 0.11 ± 0.01 l h^-1kg^-1, P < 0.05) were lower in young rats. However, when the data were reanalyzed taking body weight into consideration, the C_max (40.43 ± 3.25 vs 78.21 ± 11.23 ng kg^-1 ml^-1, P < 0.05) and AUC (182.27 ± 11.74 vs 344.62 ± 32.11 ng h^-1 ml^-1, P < 0.05) were lower in young rats. The clearance (0.03 ± 0.002 vs 0.02 ± 0.002 l h^-1 kg^-1, P < 0.05) and Vd/F (0.210 vs 0.067 l/kg) were higher in young rats. The raw data (not adjusted for body weight) strongly suggest that age plays a pivotal role in the disposition of tityustoxin. Furthermore, our results also indicate that the differences in the severity of symptoms observed in children and adults after scorpion envenomation can be explained in part by differences in the pharmacokinetics of the toxin.

Key words: Scorpion venom, Tityustoxin, Pharmacokinetics, Age, Bootstrapping resampling, Non-compartmental analysis

Introduction

Tityus serrulatus is one of most venomous scorpions. The venom of this scorpion is composed of a variety of water-soluble and -insoluble proteins among which tityustoxin (TsTX) is the most toxic component (1-3). The majority of toxins present in the venom are highly neurotoxic. The major site of action of the toxins is the sodium ion channel (4) where they modulate the release of neurotransmitters (5,6). This leads to a variety of adverse effects which include respiratory failure (7), lung edema (8,9), arrhythmias, tachycardia followed by bradycardia (10), skeletal muscle stimulation, lacrimation, convulsions, and enlarged pupils (11), among others (12,13).

A number of studies have investigated the effect of age on the toxicity of T. serrulatus scorpion venom and other scorpion venoms (2,7,8,13,14). Clot-Faybesse et al. (15) showed that 3-7-day-old mice were more susceptible to scorpion neurotoxin than 10-week-old adult mice. Similar study from our laboratory has shown that 21-22-day-old rats were more susceptible to a venom water extract administered subcutaneously (sc) than adult rats (150-160 days old) (16). These differences in toxicity have been underscored in a variety of clinical studies which have clearly shown that children exhibit a higher degree of envenomation symptoms associated with the cardiovascular and central nervous systems (17-19).

Pharmacokinetic studies of the venom of several scorpions have been reported in the literature (10,11,20-24). Santana et al. (23) have explained the disposition of scorpion venom in adult animals. The venom exhibits a rapid absorption, extensive distribution from blood to tissue and slow elimination from the organism (23). However, the influence of age and body weight on the disposition of the various toxins of scorpion venom has not been studied in detail. The present investigation was undertaken in an attempt to compare the effect of age on the disposition of TsTX in young (21-22 days old) and adult (150-160 days old) male rats.

Material and Methods

Labeling of TsTX

TsTX was obtained from T. serrulatus scorpion venom by gel filtration and ion exchange chromatography (1). The toxin concentration was estimated by the formula: protein (mg/ml) = absorbance at 280 nm/3.584. Absorbance measurements were made with a Hitachi spectrophotometer, model U-2001 (Kyoto, Japan). TsTX (200-250 µg) was labeled with 37 MBq sodium pertechnetate as described in Ref. 25. Silica gel ascending and Whatman paper descending chromatography (26) was used to monitor the labeling efficacy. ^99mTcO₂ not incorporated into the protein was retained on the Sephadex G-10 (Sigma, St. Louis, MO, USA) gel filtration column (4.0 x 1.5 cm) equilibrated with 0.9% (w/v) NaCl. Fractions 3-6 were pooled and injected ip into mice (16 µg/mouse) weighing 20-25 g (N = 4) to observe the characteristic signs of intoxication.

Blood samples for ^99m Tc-TsTX determination

Holtzman adult rats (150-160 days old) weighing 353 ± 33 g and young rats (21-22 days old) weighing 39 ± 6 g were used. The animals received water and food ad libitum. Groups of animals were injected sc with 6 µg ^99mTc-TsTX and sacrificed at 0.08, 0.5, 1, 2, 3, 6, 8 and 12 h. For sedation, all rats were injected ip with urethane (140 mg/100 g) 30 min before sacrifice by decapitation. Blood samples were collected into a tube containing ethylenediaminetetraacetic acid (EDTA) anticoagulant at a final concentration of 0.05 M. Blood aliquots (200 µl) were counted with an automatic scintillation counter (ANSR, Abbott, Chicago, IL, USA) and the results are reported as percent injected dose/ml blood. ^99mTc-TsTX (6 µg) was used as a positive control. The percent radioactivity data were reported after conversion to ng/ml blood. Animal studies were performed in accordance with the guidelines of the United Kingdom Biological Council on the use of living animals in scientific investigations.

Pharmacokinetic analysis

Groups of rats consisting of a minimum of five and a maximum of eight animals were sacrificed at 0.08, 0.5, 1, 2, 3, 6, and 12 h after sc administration of 6 µg TsTX. Due to the limitations imposed by the size of the young rats (21-22 days old), only one blood sample was collected per animal. This methodology of sample collection (one sample per animal) hampers the use of traditional methods of pharmacokinetic data analysis because the calculation of parameters like total area under the curve (AUC_0-_¥), maximum concentration (C_max) and elimination rate constant (K) requires the time-dependent collection of blood samples to obtain intra-animal concentration profiles. Consequently, an alternate method of pharmacokinetic and statistical data analysis was required which would help calculate and statistically compare the pharmacokinetic parameters obtained from "one sample per animal" data.

To overcome this problem, a bootstrapping procedure was developed using the Visual Basic feature of Excel. Bootstrap resampling is a very robust statistical method for estimating the accuracy of parameters obtained from experimental data (27). The resampling procedure generates data sets after random iterations. Resampling techniques such as bootstrapping have been shown to provide robust estimators of pharmacokinetic and pharmacodynamic parameters (28).

The program was designed to randomly select one value from a pool of concentration values at a given time point. This procedure was iterated for all the other time points to define individual concentration time profiles. The resampled data from adult and young rats were then analyzed using non-compartment analysis to obtain the mean and standard deviation of different pharmacokinetic parameters such as K using the last points from the blood concentration vs time curve, body elimination half-life (T_½K), AUC_¥, C_max, time to reach C_max (T_max), mean residence time (MRT), and clearance (CL/F). The distribution volume (Vd/F) was determined to be equal to [D/(AUC_¥ x K)], where D = 6 µg/animal, using the WINNOLIN computer program for non-compartmental analysis.

Results are reported as means ± SD or means ± SEM. The pharmacokinetic parameters obtained were analyzed by the two-tailed unpaired Student t-test, with the level of significance set at P < 0.05.

Results

The yield of radioactivity obtained after labeling the toxin was 75 to 85%. TsTX activity after labeling was intact, as determined by the observation of characteristic signs of acute scorpion intoxication in mice (piloerection, salivation, tachycardia, dyspnea, muscle contraction, and convulsion) after ip injection of the labeled toxin (16 µg/mouse).

Figure 1A and B shows the concentration-time profiles of ^99mTc-TsTX in adult and young male rats, respectively, with and without correction for body weight. When data were not corrected for body weight (Figure 1A), ^99mTc-TsTX showed a significantly higher blood concentration in young animals. C_max was reached faster by young rats while the elimination phase was apparently slower. Figure 1B, with data corrected for body weight, shows that the ^99mTc-TsTX concentration in blood at 5 and 30 min was higher in young rats, but the elimination was similar to that observed without correction for body weight.

Table 1 shows the parameters calculated from data without correction for body weight. All comparisons of parameters between the adult and young groups were significantly different. C_max was five times higher for young rats and occurred earlier than in adult rats (T_max = 0.64 ± 0.19 vs 2.44 ± 0.49 h). There was a five-fold difference in AUC_0-_¥ between the young and adult groups (296.49 ± 21.09 vs 55.96 ± 5.41 ng h^-1 ml^-1), indicating a five-fold higher systemic exposure to the toxin in young rats. T_½K and MRT were longer in young rats (T_½K = 5.00 ± 1.96 vs 2.70 ± 0.48 h, MRT = 6.77 ± 1.66 vs 4.36 ± 0.59 h). CL/F and Vd/F were higher in adult than in young rats.

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Age effects on the pharmacokinetics of tityustoxin from Tityus serrulatus scorpion venom in rats. E.A. Nunan, V. Arya, G. Hochhaus, V.N. Cardoso and T. Moraes-Santos. Brazilian Journal of Medical and Biological Research, 37 (3): 385, 2004.

Table 2 shows the values of the pharmacokinetic parameters obtained after correcting for body weight. AUC and C_max were about two times lower in young than in adult rats, but C_max was again reached earlier by young animals (T_max= 0.84 ± 0.23 vs 2.47 ± 0.51 h). The correction for body weight did not change T_½K, MRT or K values (Tables 1 and 2). CL/F and Vd/F, however, were higher in young animals after correction for body weight.

Thumbnail

Age effects on the pharmacokinetics of tityustoxin from Tityus serrulatus scorpion venom in rats. E.A. Nunan, V. Arya, G. Hochhaus, V.N. Cardoso and T. Moraes-Santos. Brazilian Journal of Medical and Biological Research, 37 (3): 385, 2004.

Figure 1.
Blood concentration-time profile of adult (circles) and young (squares) male rats after subcutaneous injection of 6 µg ^99mTc-TsTX. Data are reported as means ± SEM (N = 5-8) without correction for body weight (A) and corrected for body weight (B). *P < 0.05 compared to adult rats (Student t-test).

Discussion

A number of studies have been performed to investigate the disposition of injected TsTX. However, most of these studies have been performed on adult rats or mice. To the best of our knowledge, very few studies have investigated the disposition of TsTX in 21-22-day-old young rats (29).

In the present study, 6 µg TsTX was administered sc to young and adult rats. The dose was not adjusted for differences in body weight because, as the result of a scorpion bite, the same amount of toxin is injected, irrespective of body weight. The blood concentration versus time data were used to calculate and statistically compare the pharmacokinetic parameters. The data were analyzed with and without taking the effect of body weight into consideration. However, the pharmacokinetic parameters obtained on the basis of data not corrected for body weight should have significantly higher physiological relevance.

After the sc administration of the same amount of ^99mTc-TsTX (6 µg) the young rats showed higher C_max, higher AUC_0-_¥ and an earlier T_max, indicating a faster and higher uptake of the toxin. The increased uptake of the toxin can also be explained on the basis of a higher absorption rate for the toxin in young rats, also explaining the lower value found for T_max. As expected, the Vd/F for the uncorrected data was higher in adult rats than in young rats.

When the blood concentration versus time data were corrected for body weight, Vd/F and CL/F were increased in young rats and decreased in adult rats, inverting the relationship observed for noncorrected data. However, these parameters are strongly influenced by body surface area (30). After correcting for body weight, the Vd/F of young animals was twice that of adult animals, indicating a possible increased distribution in young rats.

The pharmacokinetic profile of TsTX found in sc injected adult rats was similar to that reported with the use of crude venom (23). The present data indicate higher and faster absorption and distribution in young rats. In spite of the limitation of the experimental protocol due to the number of points at the end of the curve, it can be said that in young rats there is a slower elimination of the toxin. This statement is based on the fact that the observed K for adult rats, which is consistent with the literature (23), was higher than for young animals.

The results indicate that pharmacokinetics can explain in part the differences in toxicity observed between children and adults after scorpion envenomation. Due to these differences in disposition of the toxin between children and adults, pharmacological interventions in children after scorpion envenomation need to be further investigated. These data are important considering that in experimental models with adult animals the pharmacokinetic data for scorpion antivenom are different from those for scorpion venom (23,31,32). Antivenom absorption is lower, as also is its distribution from blood to tissues compared to venom. Since in young rats the disposition of the toxin is modified compared to adult rats, these alterations should be investigated in terms of treatment of scorpion envenomation, especially immunotherapy.

Acknowledgments

The authors thank Maria G.V. Torquato, Alexandre Batista, and Alexsandra R. Oliveira from the Pharmacy School, UFMG, for technical assistance, and Dr. Carlos Jorge R. Simal from Felício Rocho Hospital (Belo Horizonte, MG, Brazil) for the supply of ^99mTechnetium.

Address for correspondence: T. Moraes-Santos, Faculdade de Farmácia, UFMG, Av. Olegário Maciel, 2360, 30180-112 Belo Horizonte, MG, Brasil. Fax: +55-31-3339-7666. E-mail: tmoraes@dedalus.lcc.ufmg.br

Research supported by CNPq, FAPEMIG, PRPq/UFMG, and CAPES. Received October 28, 2002. Accepted October 24, 2003.

¹
Sampaio SV, Laure CJ & Giglio JR (1983). Isolation and characterization of toxic proteins from the venom of the Brazilian scorpion Tityus serrulatus Toxicon, 21: 265-277.
2. Bucaretchi F, Bacarat ECE, Nogueira RJN, Chave A, Zambrone FAD, Fonseca MRCC & Tourinho FS (1995). A comparative study of severe scorpion envenomation in children caused by Tityus bahiensis and Tityus serrulatus Revista do Instituto de Medicina Tropical de São Paulo, 37: 331-336.
3. Kalapothakis E & Chávez-Olórtegui C (1997). Venom variability among several Tityus serrulatus specimens. Toxicon, 35: 1523-1529.
4. Massensini AR, Suckling J, Brammer MJ, Moraes-Santos T, Gomez MV & Romano-Silva MA (2002). Tracking sodium channels in live cells: confocal imaging using fluorescently labeled toxins. Journal of Neuroscience Methods, 116: 189-196.
5. Falqueto EB, Massensini AR, Moraes-Santos T, Gomez MV & Romano-Silva MA (2002). Modulation of Na⁺-channels by neurotoxins produces different effects on [³H]-ACh release with mobilization of distinct Ca²⁺-channels. Cellular and Molecular Neurobiology, 22: 819-826.
6. Nicolato R, Fernandes VMV, Moraes-Santos T, Gomez RS, Prado MAM, Romano-Silva MA & Gomez MV (2002). Release of g-[³H] aminobutyric acid in rat brain cortical slices by a-scorpion toxin. Neuroscience Letters, 325: 155-158.
7. Sofer S & Gueron M (1988). Respiratory failure in children following envenomation by the scorpion Leiurus quinquestriatus: hemodynamic and neurological aspects. Toxicon, 26: 931-939.
8. Amaral CFS, Rezende NA & Freire-Maia L (1993). Acute pulmonary edema after Tityus serrulatus scorpion sting in children. American Journal of Cardiology, 71: 242-245.
9. Mesquita MBS, Moraes-Santos T & Moraes MFD (2002). Phenobarbital blocks the lung edema induced by centrally injected tityustoxin in adult Wistar rats. Neuroscience Letters, 332: 119-122.
10. Ismail M & Abd-Elsalam A (1988). Are the toxicological effects of scorpion envenomation related to tissue venom concentration? Toxicon, 26: 233-236.
11. Ismail M, Abd-Elsalam A & Morad AM (1990). Do changes in body temperature following envenomation by the scorpion Leiurus quinquestriatus influence the course of toxicity? Toxicon, 28: 1265-1284.
12. Magalhães MM, Garbacio VL, Almeida MB, Braz ACA, Moraes-Santos T, Freire-Maia L & Cunha-Melo JR (2000). Acid-base balance following Tityus serrulatus scorpion envenoming in anaesthetized rats. Toxicon, 38: 855-864.
13. Cupo P, Jurca M, Azevedo-Marques MM, Oliveira MJS & Hering SE (1994). Severe scorpion envenomation in Brazil. Clinical, laboratory and anatomopathological aspects. Revista do Instituto de Medicina Tropical de São Paulo, 36: 67-76.
14. Amitai Y, Mines Y, Aker M & Goitein K (1985). Scorpion sting in children. Clinical Pediatrics, 24: 136-139.
15. Clot-Faybesse O, Guieu R, Rochat H & Devaux C (2000). Toxicity during early development of the mouse nervous system of a scorpion neurotoxin active on sodium channels. Life Sciences, 66: 185-192.
16. Nunan EA, Cardoso VN & Moraes-Santos T (2001). Lethal effect of the scorpion Tityus serrulatus venom: comparative study on adult and weanling rats. Brazilian Journal of Pharmaceutical Sciences, 37: 39-44.
17. Magalhães O (1938). Scorpionism. Journal of Tropical Medicine and Hygiene, 41: 393-399.
18. Freire-Maia L, Pinto GI & Franco I (1974). Mechanism of the cardiovascular effects produced by purified scorpion toxin in the rat. Journal of Pharmacology and Experimental Therapeutics, 188: 207-213.
19. Drumond YA, Couto AS, Moraes-Santos T, Almeida AP & Freire-Maia L (1995). Effects of toxin Ts-g and tityustoxin purified from Tityus serrulatus venom on isolated rat atria. Comparative Biochemistry and Physiology, 111C: 183-190.
20. Ismail M, Abdulah ME, Morad AM & Ageel AM (1980). Pharmacokinetics of ¹²⁵I-labelled venom from the scorpion Androctonus amoreuxi, Aud. and Sav. Toxicon, 18: 301-308.
21. Ismail M, Amal J, Fatani Y & Da Bees TT (1992). Experimental treatment protocols for scorpion envenomation: A review of common therapies and effect of kallikrein-kinin inhibitors. Toxicon, 30: 1257-1279.
22. Ismail M, Abd-Elsalam A & Al-Ahaidib MS (1994). Androctonus crassicauda (Olivier), a dangerous and unduly neglected scorpion. I. Pharmacological and clinical studies. Toxicon, 32: 1599-1618.
23. Santana GC, Freire ACT, Ferreira APL, Chávez-Olórtegui C, Diniz CR & Freire-Maia L (1996). Pharmacokinetics of Tityus serrulatus scorpion venom determined by enzyme-linked immunosorbent assay in the rat. Toxicon, 34: 1063-1066.
24. Calderón-Aranda ES, Rivière G, Choumet V, Possani LD & Bom C (1999). Pharmacokinetics of toxic fraction of Centruroides limpidus limpidus venom in experimentally envenomed rabbits and effects of immunotherapy with specific F(ab') 2. Toxicon, 37: 771-782.
25. Nunan EA, Cardoso VN & Moraes-Santos T (2002). Technetium-99m labeling of tityustoxin and venom from scorpion Tityus serrulatus Applied Radiation and Isotopes, 57: 849-852.
26. United States Pharmacopoeia (1995). 23rd edn. United States Pharmacopeial Convention, Inc., Rockville, MD, USA, 1312-1313.
27. Iwi GRKM, Palmer AM, Preece AW & Saunders M (1999). Bootstrap resampling: a powerful method of assessing confidence intervals for doses for experimental data. Physics in Medicine and Biology, 44: N55-N62.
28. Mager HGG (1998). Resampling methods in sparse sampling situations in pre-clinical pharmacokinetic studies. Journal of Pharmaceutical Sciences, 87: 372-378.
29. Nunan EA, Moraes MFD, Cardoso VN & Moraes-Santos T (2003). Effect of age on body distribution of tityustoxin from Tityus serrulatus scorpion venom in rats. Life Sciences, 73: 319-325.
30. Rowland M & Tozer TN (1995). Clinical Pharmacokinetics - Concepts and Applications 3rd edn. Lea & Febriger, London, UK.
31. Ismail M, Shibl AM, Morad AM & Abdullah ME (1983). Pharmacokinetics of ¹²⁵I-labelled antivenom to the venom from scorpion Androctonus amoreuxi. Toxicon, 21: 47-56.
32. Ismail M & Abd-Elsalam A (1998). Pharmacokinetics of ¹²⁵I-labelled IgG, F(ab')₂ and fractions of scorpion and snake antivenins: merits and potential for therapeutic use. Toxicon, 36: 1523-1528.

Correspondence and Footnotes

Publication Dates

Publication in this collection
20 Apr 2004
Date of issue
Mar 2004

History

Accepted
24 Oct 2003
Received
28 Oct 2002

This work is licensed under a Creative Commons Attribution 4.0 International License.

[1] ¹
Sampaio SV, Laure CJ & Giglio JR (1983). Isolation and characterization of toxic proteins from the venom of the Brazilian scorpion Tityus serrulatus Toxicon, 21: 265-277.

[2] 2. Bucaretchi F, Bacarat ECE, Nogueira RJN, Chave A, Zambrone FAD, Fonseca MRCC & Tourinho FS (1995). A comparative study of severe scorpion envenomation in children caused by Tityus bahiensis and Tityus serrulatus Revista do Instituto de Medicina Tropical de São Paulo, 37: 331-336.

[3] 3. Kalapothakis E & Chávez-Olórtegui C (1997). Venom variability among several Tityus serrulatus specimens. Toxicon, 35: 1523-1529.

[4] 4. Massensini AR, Suckling J, Brammer MJ, Moraes-Santos T, Gomez MV & Romano-Silva MA (2002). Tracking sodium channels in live cells: confocal imaging using fluorescently labeled toxins. Journal of Neuroscience Methods, 116: 189-196.

[5] 5. Falqueto EB, Massensini AR, Moraes-Santos T, Gomez MV & Romano-Silva MA (2002). Modulation of Na⁺-channels by neurotoxins produces different effects on [³H]-ACh release with mobilization of distinct Ca²⁺-channels. Cellular and Molecular Neurobiology, 22: 819-826.

[6] 6. Nicolato R, Fernandes VMV, Moraes-Santos T, Gomez RS, Prado MAM, Romano-Silva MA & Gomez MV (2002). Release of g-[³H] aminobutyric acid in rat brain cortical slices by a-scorpion toxin. Neuroscience Letters, 325: 155-158.

[7] 7. Sofer S & Gueron M (1988). Respiratory failure in children following envenomation by the scorpion Leiurus quinquestriatus: hemodynamic and neurological aspects. Toxicon, 26: 931-939.

[8] 8. Amaral CFS, Rezende NA & Freire-Maia L (1993). Acute pulmonary edema after Tityus serrulatus scorpion sting in children. American Journal of Cardiology, 71: 242-245.

[9] 9. Mesquita MBS, Moraes-Santos T & Moraes MFD (2002). Phenobarbital blocks the lung edema induced by centrally injected tityustoxin in adult Wistar rats. Neuroscience Letters, 332: 119-122.

[10] 10. Ismail M & Abd-Elsalam A (1988). Are the toxicological effects of scorpion envenomation related to tissue venom concentration? Toxicon, 26: 233-236.

[11] 11. Ismail M, Abd-Elsalam A & Morad AM (1990). Do changes in body temperature following envenomation by the scorpion Leiurus quinquestriatus influence the course of toxicity? Toxicon, 28: 1265-1284.

[12] 12. Magalhães MM, Garbacio VL, Almeida MB, Braz ACA, Moraes-Santos T, Freire-Maia L & Cunha-Melo JR (2000). Acid-base balance following Tityus serrulatus scorpion envenoming in anaesthetized rats. Toxicon, 38: 855-864.

[13] 13. Cupo P, Jurca M, Azevedo-Marques MM, Oliveira MJS & Hering SE (1994). Severe scorpion envenomation in Brazil. Clinical, laboratory and anatomopathological aspects. Revista do Instituto de Medicina Tropical de São Paulo, 36: 67-76.

[14] 14. Amitai Y, Mines Y, Aker M & Goitein K (1985). Scorpion sting in children. Clinical Pediatrics, 24: 136-139.

[15] 15. Clot-Faybesse O, Guieu R, Rochat H & Devaux C (2000). Toxicity during early development of the mouse nervous system of a scorpion neurotoxin active on sodium channels. Life Sciences, 66: 185-192.

[16] 16. Nunan EA, Cardoso VN & Moraes-Santos T (2001). Lethal effect of the scorpion Tityus serrulatus venom: comparative study on adult and weanling rats. Brazilian Journal of Pharmaceutical Sciences, 37: 39-44.

[17] 17. Magalhães O (1938). Scorpionism. Journal of Tropical Medicine and Hygiene, 41: 393-399.

[18] 18. Freire-Maia L, Pinto GI & Franco I (1974). Mechanism of the cardiovascular effects produced by purified scorpion toxin in the rat. Journal of Pharmacology and Experimental Therapeutics, 188: 207-213.

[19] 19. Drumond YA, Couto AS, Moraes-Santos T, Almeida AP & Freire-Maia L (1995). Effects of toxin Ts-g and tityustoxin purified from Tityus serrulatus venom on isolated rat atria. Comparative Biochemistry and Physiology, 111C: 183-190.

[20] 20. Ismail M, Abdulah ME, Morad AM & Ageel AM (1980). Pharmacokinetics of ¹²⁵I-labelled venom from the scorpion Androctonus amoreuxi, Aud. and Sav. Toxicon, 18: 301-308.

[21] 21. Ismail M, Amal J, Fatani Y & Da Bees TT (1992). Experimental treatment protocols for scorpion envenomation: A review of common therapies and effect of kallikrein-kinin inhibitors. Toxicon, 30: 1257-1279.

[22] 22. Ismail M, Abd-Elsalam A & Al-Ahaidib MS (1994). Androctonus crassicauda (Olivier), a dangerous and unduly neglected scorpion. I. Pharmacological and clinical studies. Toxicon, 32: 1599-1618.

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