- Email: [email protected]
Distribution of 125I labelled scorpion (Leiurus quinquestriatus H & E) venom in rat tissues
Toxicon, 1974, Vol. 12, pp . 209-211 . PoWmon Press. Printed in Great Britain
SHORT COMMUNICATION DISTRIBUTION OF 125 1 LABELLED SCORPION
(LEIUR US Q UINQ UESTRIATUS H & E) VENOM IN RAT TISSUES M. ISMAIL, * GABRIELLA K.EitTEsz,t O. H. OSMAN and M. S. SmRA Department of Pharmacology and Radioisotope Unit, Department of Pathology, University of Khartoum, Khartoum, Sudan (Acceptedfor publication 10 August 1973)
THE PRINCIPAL effects of envenomation by scorpion Leiurus quinquestriatus venom consist of hypertension, respiratory failure and skeletal muscle stimulation (MOHAMMED, 1942, 1950; ADAM and WEIss, 1959 ; ADAM et al., 1966; ISMAIL et al., 1972) . The venom also stimulates a variety of smooth muscles (MOHAMMED, 1950; OSMAN etal., 1972) and produces hyperthermia in rabbits when injected intraventricularly (OSMAN et al., 1973). The present
work was initiated with the purpose of studying the distribution of the venom in the various organs of the rat using 1251 labelled venom and to find out whether or not a correlation exists between the concentration of the venom in the organs examined and its pharmacological activity. Venom was obtained from mature L. quinquestriatus by electrical stimulation of the telson as described by ADAM et al. (1966) . Radioiodination of the venom was carried out using an adaptation of the method of ROSA et al. (1965). The method utilizes the reaction between the venom protein and nascent labelled 12 which attacks the amino acids of the venom, mainly tyrosine . Twenty mg of the lyophilized venom were dissolved in 5 ml of 0"9 per cent sodium chloride solution containing 2-5 x 10 -4 M potassium iodide (pH 7"4) and placed in the anodic space of an electrolytic cell . One half mCi of 1251 in the iodide form free from reducing agents was placed in the cathodic space, which was separated from the anodic space by a semipermeable membrane . Electrolysis was carried out for 60 min using a current strength of 2 V and applying 300 pA/tna2. Unbound iodine was removed by dialysis against 500 ml of 0-9 per cent sodium chloride solution with 6 changes of the salt solution over a period of 36 hr. The specific activity of the labelled venom was 7-5 lsCi/mg. Electrophoretic separation of the iodinated and non-iodinated dialysed venom was carried out in acetate buffer, pH 4-2, as described by EL-ASMAR et al. (1972) . The two venoms showed identical electrophoretic behaviour. Autoradiography of the electrophoretic runs showed that the radioactivity was distributed in all fractions of the iodinated venom. The venom (0"5 mg per kg) was injected either intramuscularly or intraperitoneally into female albino rats (150-200 g). This dose was previously shown to kill all rats within 1-2 hr (Ismail and Osman, unpublished observation). The labelled venom was of the same *Visiting lecturer, Department of Pharmacology, Faculty of Pharmacy, University of Abxandria, Fßypt. tPresent address, Department of Medicine, University Medical School, Debrecen, Hungary. TOXICON 1974 VoL 12
209
210
M. ISMAIL, GABRIELLA KERTESZ, O. H. OSMAN and M. S. SIDRA
lethality as the dialysed non-labelled venom. Control rats were injected with dialysed nonlabelled venom (0-5 mg per kg intramuscularly) in one leg and 1231 (3-75 ILCi per kg intramuscularly) in the other leg. All rats were killed 1 hr after injection of the venom. Samples of blood were collected on weighed stainless steel planchets. Other organs were dissected, freed from extraneous tissues, washed with saline, blotted between filter papers, transferred to stainless steel planchets and weighed. The tissues were dried in an oven at 110°C for 60 min and then transferred to test tubes. Activity measurements were carried out in this solid state using a thin-walled well type NaI crystal connected with a Packard Selektronik one-channel analyser. Radioactivity was expressed as counts per min per g wet weight . The distribution of the venom in the various organs of the rat was the same whether the venom was injected intramuscularly or intraperitoneally. Similarly, when pooled homogenates of the different tissues in 0-9 per cent saline were used, or when the proteins were precipitated with trichloroacetic acid, dissolved in 0-05 N NaOH and hyamine and counted in a well type scintillation spectrometer, the pattern of distribution of radioactivity was the same as that obtained by the drying method . As can be seen from Fig. 1, the highest radioactivity was found in the kidney and the lowest radioactivity was found in the brain.
a P ô 3 0
3
11, e
7000 6000 5000 4000
É
3000
c
2000
0 U
pl.obeled venom group =Control group
1000 0
FYo. 1. Dg1RIatmoN of 1 '°I , "_as, " w L. grdnqueatriatus vEmlsr na RAT T18suEs. B, blood; Br, brain; D, diaphragm; H, heart ; K, kidney ; Li, liver; Lu, lung ; M, muscles; S, spleen ; T, thymus ; U, uterus. Doses of the labelled venom, dialysed venom and 12-11 as in text. Values are the average of 5 observations . The vertical lines indicate 5.ß.M. At the asterisks, values are significantly different from control values (P<0-05).
Varying degrees of radioactivity were found in the blood, lungs, heart, liver, diaphragm, uterus, thymus, spleen and muscles, but of all these tissues only the kidneys, blood and lungs showed significantly higher radioactivity than the control tissues (P < 0-05). The high radioactivity of the tissues appears to be due to the labelled venom and not to the iodine liberated from it, since much less activity was found in control rats injected with both the non-labelled venom and 1231 in the same dose as the labelled venom. A factor in favour of this assumption comes from measurements of the radioactivity in the thyroid glands of both the control and labelled venom groups where much less activity was found in the latter group. The labelled venom group had a value of 316 f 38 counts per min per mg wet weight whereas that of the control group was 6365 f 855 counts per min per mg wet weight (P<0-005). The high radioactivity found in the kidneys is probably due to the rapid rate of excretion of the labelled venom (or labelled venom fractions) since high radioactivity was TDZlCON 1974 Vol. 12
Distribution of 1251 Scorpion Venom
211
also found in the urine 15 min after injection. The urine of the control group, on the other hand, contained much lower radioactivity when compared with the labelled venom group. The low radioactivity in the brain correlates well with the assumption that L. quinquestriatus venom under normal conditions does not easily penetrate the blood brain barrier (OsMAN et al., 1973). The high radioactivity in the lungs and heart may provide an explanation for the finding that these organs are among the primary sites for the action of the venom. REFERENCES ADAM, K. R. and WEtss, C. (1959) Action of scorpion venom on skeletal muscle. Br. J. Pharrnac . 14, 334. ADAM, K. R., SmEwmT, H., STAmnu, R and Wmss, C. (1966) The effect of scorpion venom on single myelinated now fibres of the frog. Ar. J. Pharmac. 26, 667. Et-An mt, M. F., ImmmN, S. A. and RAEE, F. (1972) Fractionation of scorpoin (Leiurus quinquestriatus H do E) venom. Toxicon 10, 73. IsMAH, M., OwAN, O. H., IenAww, S. A. and EL-AsmAR, M. F. (1972) Cardiovascular and respiratory responses to the venom from the scorpion Leiurw qubiquestriatus . E. Afr. med. J. 49, 273. MOHAMMED, A. H. (1942) Preparation of antiscorpion serum, use of atropine and ergotoxine. Lancet 2,364. MOHAMMED, A. H. (1950) Pharmacological action of the toxin of the Egyptian scorpion . Nature, Land. 166, 734. OsmAN, O. H., LvAmL, M ., EUAswut, M. F. and ImtAmm, S. A. (1972) Effect on the rat uterus of the venom from the scorpion Leturus qubiquratriatus. Toxican 10, 363. OSMAN, O. H., IsMAtt, M. and WeNtoEte, T. (1973) Hyperthermic response to intraventricular injection of scorpion venom : role of brain monoamines. Toxicon 11, 361 . ROSA, U., ScAssuAli, G., Pm*an, F., AmRosnao, F., Lnv.AToat, J., FEDERiarn, G., DoNATo, L. and BUNcH4 R. (1965) Protein radioiodination by an electrolytic technique. Sondoband Strahientherapie 60, 258.
TOYICON 1974 Vol. 12
SHORT COMMUNICATION DISTRIBUTION OF 125 1 LABELLED SCORPION
(LEIUR US Q UINQ UESTRIATUS H & E) VENOM IN RAT TISSUES M. ISMAIL, * GABRIELLA K.EitTEsz,t O. H. OSMAN and M. S. SmRA Department of Pharmacology and Radioisotope Unit, Department of Pathology, University of Khartoum, Khartoum, Sudan (Acceptedfor publication 10 August 1973)
THE PRINCIPAL effects of envenomation by scorpion Leiurus quinquestriatus venom consist of hypertension, respiratory failure and skeletal muscle stimulation (MOHAMMED, 1942, 1950; ADAM and WEIss, 1959 ; ADAM et al., 1966; ISMAIL et al., 1972) . The venom also stimulates a variety of smooth muscles (MOHAMMED, 1950; OSMAN etal., 1972) and produces hyperthermia in rabbits when injected intraventricularly (OSMAN et al., 1973). The present
work was initiated with the purpose of studying the distribution of the venom in the various organs of the rat using 1251 labelled venom and to find out whether or not a correlation exists between the concentration of the venom in the organs examined and its pharmacological activity. Venom was obtained from mature L. quinquestriatus by electrical stimulation of the telson as described by ADAM et al. (1966) . Radioiodination of the venom was carried out using an adaptation of the method of ROSA et al. (1965). The method utilizes the reaction between the venom protein and nascent labelled 12 which attacks the amino acids of the venom, mainly tyrosine . Twenty mg of the lyophilized venom were dissolved in 5 ml of 0"9 per cent sodium chloride solution containing 2-5 x 10 -4 M potassium iodide (pH 7"4) and placed in the anodic space of an electrolytic cell . One half mCi of 1251 in the iodide form free from reducing agents was placed in the cathodic space, which was separated from the anodic space by a semipermeable membrane . Electrolysis was carried out for 60 min using a current strength of 2 V and applying 300 pA/tna2. Unbound iodine was removed by dialysis against 500 ml of 0-9 per cent sodium chloride solution with 6 changes of the salt solution over a period of 36 hr. The specific activity of the labelled venom was 7-5 lsCi/mg. Electrophoretic separation of the iodinated and non-iodinated dialysed venom was carried out in acetate buffer, pH 4-2, as described by EL-ASMAR et al. (1972) . The two venoms showed identical electrophoretic behaviour. Autoradiography of the electrophoretic runs showed that the radioactivity was distributed in all fractions of the iodinated venom. The venom (0"5 mg per kg) was injected either intramuscularly or intraperitoneally into female albino rats (150-200 g). This dose was previously shown to kill all rats within 1-2 hr (Ismail and Osman, unpublished observation). The labelled venom was of the same *Visiting lecturer, Department of Pharmacology, Faculty of Pharmacy, University of Abxandria, Fßypt. tPresent address, Department of Medicine, University Medical School, Debrecen, Hungary. TOXICON 1974 VoL 12
209
210
M. ISMAIL, GABRIELLA KERTESZ, O. H. OSMAN and M. S. SIDRA
lethality as the dialysed non-labelled venom. Control rats were injected with dialysed nonlabelled venom (0-5 mg per kg intramuscularly) in one leg and 1231 (3-75 ILCi per kg intramuscularly) in the other leg. All rats were killed 1 hr after injection of the venom. Samples of blood were collected on weighed stainless steel planchets. Other organs were dissected, freed from extraneous tissues, washed with saline, blotted between filter papers, transferred to stainless steel planchets and weighed. The tissues were dried in an oven at 110°C for 60 min and then transferred to test tubes. Activity measurements were carried out in this solid state using a thin-walled well type NaI crystal connected with a Packard Selektronik one-channel analyser. Radioactivity was expressed as counts per min per g wet weight . The distribution of the venom in the various organs of the rat was the same whether the venom was injected intramuscularly or intraperitoneally. Similarly, when pooled homogenates of the different tissues in 0-9 per cent saline were used, or when the proteins were precipitated with trichloroacetic acid, dissolved in 0-05 N NaOH and hyamine and counted in a well type scintillation spectrometer, the pattern of distribution of radioactivity was the same as that obtained by the drying method . As can be seen from Fig. 1, the highest radioactivity was found in the kidney and the lowest radioactivity was found in the brain.
a P ô 3 0
3
11, e
7000 6000 5000 4000
É
3000
c
2000
0 U
pl.obeled venom group =Control group
1000 0
FYo. 1. Dg1RIatmoN of 1 '°I , "_as, " w L. grdnqueatriatus vEmlsr na RAT T18suEs. B, blood; Br, brain; D, diaphragm; H, heart ; K, kidney ; Li, liver; Lu, lung ; M, muscles; S, spleen ; T, thymus ; U, uterus. Doses of the labelled venom, dialysed venom and 12-11 as in text. Values are the average of 5 observations . The vertical lines indicate 5.ß.M. At the asterisks, values are significantly different from control values (P<0-05).
Varying degrees of radioactivity were found in the blood, lungs, heart, liver, diaphragm, uterus, thymus, spleen and muscles, but of all these tissues only the kidneys, blood and lungs showed significantly higher radioactivity than the control tissues (P < 0-05). The high radioactivity of the tissues appears to be due to the labelled venom and not to the iodine liberated from it, since much less activity was found in control rats injected with both the non-labelled venom and 1231 in the same dose as the labelled venom. A factor in favour of this assumption comes from measurements of the radioactivity in the thyroid glands of both the control and labelled venom groups where much less activity was found in the latter group. The labelled venom group had a value of 316 f 38 counts per min per mg wet weight whereas that of the control group was 6365 f 855 counts per min per mg wet weight (P<0-005). The high radioactivity found in the kidneys is probably due to the rapid rate of excretion of the labelled venom (or labelled venom fractions) since high radioactivity was TDZlCON 1974 Vol. 12
Distribution of 1251 Scorpion Venom
211
also found in the urine 15 min after injection. The urine of the control group, on the other hand, contained much lower radioactivity when compared with the labelled venom group. The low radioactivity in the brain correlates well with the assumption that L. quinquestriatus venom under normal conditions does not easily penetrate the blood brain barrier (OsMAN et al., 1973). The high radioactivity in the lungs and heart may provide an explanation for the finding that these organs are among the primary sites for the action of the venom. REFERENCES ADAM, K. R. and WEtss, C. (1959) Action of scorpion venom on skeletal muscle. Br. J. Pharrnac . 14, 334. ADAM, K. R., SmEwmT, H., STAmnu, R and Wmss, C. (1966) The effect of scorpion venom on single myelinated now fibres of the frog. Ar. J. Pharmac. 26, 667. Et-An mt, M. F., ImmmN, S. A. and RAEE, F. (1972) Fractionation of scorpoin (Leiurus quinquestriatus H do E) venom. Toxicon 10, 73. IsMAH, M., OwAN, O. H., IenAww, S. A. and EL-AsmAR, M. F. (1972) Cardiovascular and respiratory responses to the venom from the scorpion Leiurw qubiquestriatus . E. Afr. med. J. 49, 273. MOHAMMED, A. H. (1942) Preparation of antiscorpion serum, use of atropine and ergotoxine. Lancet 2,364. MOHAMMED, A. H. (1950) Pharmacological action of the toxin of the Egyptian scorpion . Nature, Land. 166, 734. OsmAN, O. H., LvAmL, M ., EUAswut, M. F. and ImtAmm, S. A. (1972) Effect on the rat uterus of the venom from the scorpion Leturus qubiquratriatus. Toxican 10, 363. OSMAN, O. H., IsMAtt, M. and WeNtoEte, T. (1973) Hyperthermic response to intraventricular injection of scorpion venom : role of brain monoamines. Toxicon 11, 361 . ROSA, U., ScAssuAli, G., Pm*an, F., AmRosnao, F., Lnv.AToat, J., FEDERiarn, G., DoNATo, L. and BUNcH4 R. (1965) Protein radioiodination by an electrolytic technique. Sondoband Strahientherapie 60, 258.
TOYICON 1974 Vol. 12