- Email: [email protected]
Pharmacological studies of snake (Bitis arietans) venom
TUaie+w~. 1974,
Yot.
17, Op. 169-373. t~apmoo Pra Teino~d la üert Drhaie .
PHARMACOLOGICAL STUDIES OF SNAKE (B1TIS ARIETANS) VENOM O. H. 05C+uIV and K. A. GuM~A Departments of Pharmacology and Biochemistry, University of Khartoum, Sudan (~lccepred for publication 23 btay 1974) O. H. Osr4rty and K. A. Gtn~tM . Pharmacological studies of snake (Bins arlerans) venom. Toxkwr 12, 369-373, 1974 .-Venoun from Bins arierans was fractionated on Sephadea G-100 into flue main fractions, of which only fraction 1 was lethal to mice . The crude venous and fractions 1 and 2 produced a gradual and precipitous fall in the blood pressure of the cat . This fall was not antagonised by prctrsatment with hexamethonium, cyproheptadine, propranobl, or atropine . Also, it was not blocked by indomethacin or the kinin inhibitor, trasylol . Both the crude venom and fraction 1 produced a depression of the isolated bean which was not prevented by atropine or hexamethonium. Similarly, the crude venom and fractions 1 and 2 increased the capillary permeability in rabbits. Thecrude venom and fractions 1 and 3 pradrrced an inhibition of a variety of smooth muscle preparations such as the rabbit intestine, guineapig ileum and the rat uterus . However, the spontaneously contracting rat uterus was stimulated by the crude venom and fraction I. The venom produced hyperglycaemia in the rabbit and a decrease in the potassium level of the cat blood. It is suggested that the circulatory shock produced by the venom is due to a direct inhibition of vascular smooth muscle, together with a direct dcpr+casion of the myocardium and an increased capillary permeability . INTRODUCTION
Bltls arletans, the puff' adder, is one of the commonest and most widespread of African snakes (BROADLY, 1968). It has been claimed to be responsible for most cases of snake bite in Central and Southern Africa (C1IAPMAN, 1968) . Its venom was said to be primarily cytotoxic (CHAeMAN,1968), and to produce both local and genernlized internal haemorrhagic lesions (CHRt3TFNSEN, 1968). GnASSE~r et al. (1956) had fractionated the venom into 4 or 5 components by the use of paper electrophoresis, all of them migrating towards the anode. The crude venom consists of a complex mixture of proteinases and amino acid esterases (DELPIERRE, 1968) . Gtrrtat et al. (1963) found that the venom also contained copper. The Ln~ of the crude venom was determined by Fttt:YVOCt=.I. (1965) in mice to be 1-2 ltg per g when injected subcutaneously . The main pharmacological effects of the crude venom are hypotension, and bradycardia (Vtctc et al., 1967), collagenolytic activity, anticoagulant activity (Ctttt.tsTExserr, 1968), and inhibition of mitochondria) respiration (TAUB and Er.uorr, 1964) . The present study was carried out with the aim of examining in some detail the pharmacological properties of the crude venom and some of its fractions on different isolated tissues and animal preparations. MATERIALS AND METHODS
Lyophilized Bias arietans venom was obtained from Sigma Chemical Company, St. Louis, Missouri, U.S.A. Fractionation of the crude venom was performed using Sephadex G-100 (Sigma) column with bed dimension of2~5 x 35 cm equilibrated with 001 M phosphate buffer, pH 7~4. Twenty milligrams of dried venom dissolved in 3 ml of phosphate buffer roxrcox t9m r o r. u
369
370
O. EL 06MAN and IL ti GIJMM
wtre applied to the column. Flution was continued with 001 M phosphate buffer and 5 ml fractions were collected at flow rates of 35-40 ml per hr. Protein was estimated spectrophotometrically at 280 nm. Solutions of the crude venom or the fractions wen kept at 4°C until used. Blood pressure recording was carried out in cats (IseutL et al., 1972). The isolated heart (Langendorfl) preparation was performed using rabbits (Os~N et al., 1973). The assessment of vascular permeability was carried out by an adaptation of the method of Jtrn~t and WILLOUGHHY (1962) . The abdominal skin of adult albino rabbits was shaved and 24 hr later the animals were injected i.v. with Evans blue dye (7 mg per kg). The crude venom or the fractions were injected i.d. on both sides of the midline of the shaved area. Twenty minutes later, the estimations of the dye leakage were made by the macroscopic appearance of the skin . Blood glucose was determined according to the method of MARKS (1959). Rabbits of either sex weighing between 1"5 and 2 kg were fasted overnight. The venom was injected i.v. in a dose of 300 ~g per kg. Blood samples were withdrawn from a marginal ear vein before and at 30 min intervals aRer injection of the venom for a periôd of 2 hr. Sodium and potassium were determined in cats using whole blood rather than serum in order to avoid errors that might arise as a result of the haemolytic effect of the venom. Both sodium and potassium were determined by the flame photometer method . Cats of either sex weighing between 2 and 4 kg were anaesthetized with pentobarbitone sodium (40 mg per kg i.p.). The venom was injected i.v. in a dose of 300 ~g per kg through a femoral vein. Blood samples were taken from a common carotid artery before and at } hr intervals over a period of 3 hr after injection of the venom. The venom and its fractions were tested on the following preparations : the isolated rabbit intestine (MAGNUS, 1904), guinea-pig ileum, the isolated pcriartcrially stimulated rabbit jcjenum (FINKGLMAN, 1930), the isolated rat uterus (Dt: JALOIY et al., 1945), the spontaneously contracting rat uterus in Ringer-Locke solution ('To'rt11LL, 1965), and the isolated phrenic ncrvo-hcmidiaphragm of the rat (BGLaRtHa, 1946). The following drugs were used : acetylcholine chloride (Roche), atropine sulphate (The British Drug Houses Ltd.), cyproheptadine (Merck Sharp & Dohme), hcxamcthonium chloride (Fluka), histamine acid phosphate (British Drug Houses), indomethacin (Merck Sharp & Dohme), phenoxybenzamine hydrochloride (Smith Kline & French), propranolol (LC.L), serotonin creatinc sulphate (British Drug Houses), and trasylol (5000 kallikrein inactivation units per ml, Bayer). 1tFSULTS
Fractionation of the crude venom of Billsarietans by Sephadex G-100 revealed 5 fractions (Fig. 1). Only fraction 1 was lethal to mice when injected in a dose of 1 ~g per g i.p. Shortly after injection there was motorexcitement and restlessness which was followed by depression and finally convulsion and death 1-2 hr later. Autopsy revealed haemorrhage in the lungs and kidneys. The other four fractions were non-toxic when given in doses between 05-1 ~g per g i.p. No peculiar behaviour or symptoms were observed . Eß"ect ojtbe
venom on
arterial blood pressure (Fig. 2)
Intravenous injection of the crude venom in doses of 50-300 ~g per kg produced after a latent period of 10-20 sec a gradual and marked fall in the blood pressure of anaesthetized cats that persisted for 30 min or more and then returned to near-normal levels . Repeating TOXICON I971 Vd. l1
Bi~ir arietana Venom
,7i .
571
..
~n
1
;n -
t
I `ubR na
~u
f~~
I~" "ti ~. sv nv
(eaGh S ml
FI(F . I . FRACTIONATION OF 2O mti B. aI1C1!!!!S VF;NOM ON SEPIIADEX G~I00 COLUMN ; O'OI PF105PHATE SUFFCR, pFl 7-4 . vras 280 nm .
Protein
M
cstimatcd spoctrophutumctrically at
the injection of the venom caused a further fall in blood pressure that reached a very low level and resulted in circulatory shock :tnd death of the animals, Both fr :tctian I and 2 produced hypotension when injected in doses of 25-SU Ng per kg. however, when cyual conccntrttions were used, fraction 2 was more potent than either fraction I or the crude venom. Other fractions did not produce any significant etï'~~a on the arterial blood pressure . The hypotensive elfcct of the venom or its fractions were not antagonized by pretreatment with Itexamcthottium (7 mg peT kg), cyproheptadine (2 mg pc;r kg), propranolol (0-5 mg per kg), atropine (2 mg per kg), indomethacin (25 mg per kg), or trasylul (l ml per kg) . The venom produced a degrease in the rate and depth of respiration which was transient and returned to normal despite the fall in blood pressure. Usually arrest of respiration was preceded by circulatory arrest . E,Eject of the venom on the isolated rabbit heart ( Fig . 3) The crude venom in doses of 50-100 pg produced a marked decrease in both the force and rate of cardiac contraction . This cardiac depressing activity was not blocked by atropine (3001tg), or hexamethonium (l mg) . Fraction l also produced a very conspicuous depressant elTect on the heart but fraction 2 was less elFective in this respect. The other fractions were without activity. Effect of the venom on capillary permeability The crude venom in doses of 50-100 ~g per kg produced an increase in capillary permcabi(ity in the rabbit as assessed by the degree of dye leakage into the skin 20 min after injection of the venom. The average diameter of the blue area (Mean f S.E.) obtained from TOXICON 197 : Yal. ! :
372
O. H. 05MAN and K. A. GUMAA
mmHg 140
loo so Zo
mmHg loo so
r~
20
. (d)
. (e)
"
(f)
" ~(g)
FIU. 2. TIIE LFFFt"r of ~. Qr1l'i(III.K VF~~t on 7H1: CAr OLOt1P PRF:StiURE,
'The vcltunt was injected at A, li, C, U, t:, F and G (IW lag per kgl. Flcxmethonium, 7 mg per kg was injected 70 min txfurc li . Cypn~hcptadine, 2 mg per kg was injected 10 min lx:fun: C . Propranulol, 0~5 mg per kg was injected l0 min before f) . Atropinr, 2 mg per kg was injatcd 10 min before E. Indultxxthacin, 25 mg per kg was injected i.p . 30 min before F . Trnsylul, l ml per kg was injected 20 min before G .
the vcnum-treated animals was 24 ~: l'I mm as compared to 4 -~ 007 mm obtained from the saline-treated controls. Six rabbits were used in each test, Extensive oedema was still present at the site of injection several days after the venom injection . Similarly, both fractions l and 2 produced extensive capillary permeability when injected in doses of 20 pg per kg. Fraction 2 was much more potent in this respt:ct. The average diameter of the bloc area in mm (Mean ~ S.E.) obtained from 4 animals each for fractions 1 and 2 were 27 f 2'0 mm and 31 ~ l'S mm, respectively. The capillary permeability produced by the venom or the fractions was not modified by pretreatment with either cyprohcptadine (1 mg per kg), trasylol (1 mg per kg) or indomethacin (25 mg per kg). E.(ject of !!te t"enom on isolalel! smo~~th musc% preparaJiorrs The pendular movement of the isolated rabbit intestine was significantly inhibited by the crude venom (5-10 )tg per ml) . Only fraction 3 caused an inhibition of the pendular movement of tht: intestine in doses of 1-2 pg per ml. Other fractions were inclFcctivc or produced slight inhibition (Fig. 4). This inhibitory elTcct was not blocked by phenoxybenzamine (1 Itg per ml) and/or propranolol (l Ng per tnl) . TOXICO~V 1971 F'ul. !:
min
(a)
(b)
(c)
(d)
(e)
4 r
cm (a)
(b)
(c)
(d)
(e)
(f)
3 . ~Î111 : I:FFFCy u( />i. arirluus VENUh1 ()N THE IS()LATF.U RAHHITS HEART . The venom w ;as injecteJ at (a), (hl anJ (c) in a J(rc uf SU Ftg pcr hcart . At (J), fraction I was injmtml (25 lagl . At (ch fra(aion 3 was injcctcJ ('_5 lagl . Atropine, 3(1(1 lag was injectcJ IU min Ixfurc (hl . Hexantethuniunt, t mg wa+ injmtcJ 10 min befurc (c) . F~Ir : .
hIC: . -( . TI1l. rFFE(T OF
R. urirlune
VF:NIIM uN '111L PLNUULAR MUVEMF.Nr OF TIIE RAültfr~ti 1 N~rF 3 rl NE .
At (al, R. r)rirrmr venom IU lag per ml was aJJeJ tu the bath . At (h), fraction I (5 Elg per n)1) ; Ic), fr;tction Z (5 lag per ml) ; (J1, frac(iult 3, (3 lag per ntll : (el. fraction 4, (5 !lg per mll anJ If 1, fraction S, (S lag per ntl> were aJJcJ . Bath vohlnte, 25 ml .
ro .rico.v ry-.r t-.,1 .
1:
r.,,.
sr_
r
crn
F'II, . $. I~III~ F11~1('~1 (+I I1. ((r7l'1(lll.+' Y'l .!~(HI (+\ IIII . tiIKIN~f "1til~++l1":LY' ('(IVIKAI'l'Itrli KA~r 111~I~Kl ;ti. Ital utrru, in ocstrua, hath culunx: III ntl, tcntl>Lralurc 37 C-.At (a), R.urirtune acnom,2(lug Ixr ntl, vca+ aililcJ to thc hath . At (.h), fraction I (10Icg Ixr ntl) aml at fcl, fraction ' (10 y,g Ixr ml) wcrc ad~lcd .
lm .rrcm.v 14v Iv,l. 1_
BItU nrietanr Veaom
373
Both acetylcholine (0~2 ltg per ml) and histamine (0~1 Irg per ml) induced contractions on the guinea-pig ileum preparation which were inhibited molt: than 80 per ant by concentrations of the crude venom ranging between 5 and 10 ~g per ml. Similar results were obtained on the rat uteruu preparation in de Jalon solution when 5-hydroxytryptamine (0" 1 Ng per ml) was used as the agonist. Fraction 1 (5 pg per ml) completely antagonized histamine contractions on the guinea-pig ileum but it had little activity against acetylcholine induced contractions, while fraction 3 mainly antagonized atxtylcholine . Fractions 2, 4 and 5 were inactive. The crude venom (20 Itg per ml) and fraction 1 (10 ~g per ml) produced a stimulant activity on the spontaneously contracting rat uterus in Ringer-Locke solution ; the frequency and amplitude of contraction being increased (Fig. S). Fraction 2 produced a slight effect, while other fractions had no effect on this preparation . Neither the crude venom, nor any of its fractions had any effect on adrenergic transmission as evident from their effect on the periarterially stimulated rabbit jejenum. Also, they had no action on neuromuscular transmission as evident from their effect on the isolated phrenic nerve-hemidiaphragm ofthe rat . EJfec~ of the venom on blood glucose, sodium and potassirun Intravenous injection of the crude venom (300 Itg per kg) into fasting rabbits produced a significant rise in blood glucose level which reached its maximum 1 hr after injection and then began to return to normal level by the end of a 2 hr period . Similarly, when the venom was injected i.v . (300 4tg per kg) in anaesthetized cats, it produced a significant decrease in potassium level of the whole blood that reached its maximum 2 hr after injection and gradually cost again to prcinjcction Icvcl after a 3 hr period . Sodium levels however, were not significantly alTected (Table 1). TASLe l . EFFecr OF
Gluoose (mg/100 mp Na* (mequiv./I) K" (mequiv./l)
0 93 t 4ß (6)' 144 t 6~3 (3) 40~3 t 1~1 (3)
Buts artaanr veNOw, (300 ~g per kg i.v.) IN RAaaR3 AND Na+ AND K* LëVE[-t IN 30 128t f 6~4 (ti) 143 f 6~2 (3) 30t f 32 (3)
Blood levels aRer Injection 60 90 120 I32t 133t 109t t t t 6~2 4~2 3~2 (t7 (6) (6) 143 140 I4l t f f 3~1 8~9 10~6 (3) (3) (3) 26~6t 23~6t 23~8t f f f 1-0 1~3 0~33 (3) (3) (3)
ON BLOOD OLUCOÔE LEVEL CATS
130 100 t 3~9 (6) 142 f 11~6 (3) 32~5 f 3~8 (3)
" lagures in parentheses indicate the number of animals used. Results arc presented as means f standard error. fi Values are :ignificantly different from the oontmls (P<0-03). DISCUSSION
180 98 f 4~3 (6) 142 t 11~6 (3) 33 f 1~7 (3)
(min)
The principal clinical effects of envenomation by viper venom are local and generalized internal haemorrhagic lesions which may be so severe as to cause immediate death (CIIRISTGNSEN, 1968). The present studies show that Bibs arfetans venom could be fractionroxicoN t9~~ vor. r:
S74
O. H . 05MAN and K. A. GLT1VtM
ated on Sephadex G-100 into 5 main fractions. This is in agreement with the results of Ga~~ et al. (1956), who used paper electrophoresis. Only fraction 1 was lethal when injected into mice, and produced haemorrhagic lesions in both the lungs and kidneys. It is evident from the experimental results that at least three factors arecontributing to the circulatory shock caused by the venom or its fractions. Firstly, a direct inhibitory effect of the vascular smooth muscles resulting in vasodilatation of the arterioles. This effect was notantagonized by ganglionic blocking agents, antihistaminic, antiserotonergic, ß-adrenergic receptor blocking drugs or cholinergic blockers. Similarly, it was not counteracted by drugs which inhibit prostaglandin or kinin actions such as indomethacin and trasylol . Secondly, we observed a direct depressant action on the myocardium, and thirdly, an extensive increase in vascular permeability that was not modified by pretreatment with either antihistamines, antiserotonergics, or agents which inhibit kinins or prostaglandins . Vtctc et al. (1967), using dogs, showed that evisceration did not prevent the hypotensive effect of Bibs arietans venom but eliminated the bradycardia. They suggested that the venom did not produce a marked pooling of blood in the hepato-splanchnic bed ; however, pulmonary pooling might be responsible for the hypotension. It has been shown by 7.~xt et al. (1970) that the increase in capillary permeability produced by Echis carinatus venom or one of its fractions was prevented by pretreatment with the antihistaminic drug promethazine . In the present studies, however, promethazine was ineffective in antagonizing either the hypotension or the increase in capillary permeability produced by Bitis arietans venom or its fractions (unpublished observations). It is known that Bitis arierans venom contains a complex mixture of proteinases and amino acid esterases (Decr~exRe,19G8), and these enzymes may produce some breakdown products in the body such as adenosine, lysolecithin, certain fatty acids, and histamine which may have deleterious clTe~cts on the heart and circulation (CFIRISTIiNSCN, 1968). The effect of the crude venom and some of its fractions on smooth muscles scans to be generally inhibitory in nature and directly mediated. The spontaneously contracting rat uterus preparation is a notable exception, since both the crude venom and fraction L increased the frequency and amplitude of uterine contraction . This stimulant effect was not antagonized by either atropine, hcxamcthonium or mcthysergide but was partially blocked by indomethacin indicating that the effect may be mediated through release of prostaglandins and/or kinins (unpublished observations). Neither the venom nor any of its fractions affected adrenergic transmission or neuromuscular transmission of skeletal muscles. The hyperglycaemic effect of the venom may be due to a stress reaction resulting in the liberation of catecholamines from the adrenal medulla; however, this needs confirmation . Potassium levels of the whole blood were decreased by about 41 per cent 2 hr after injection of a sublethal dose of the venom. Sodium levels, however, were not significantly changed. Potassium lowering may be due to the increased release of ACTH from the anterior pituitary gland (MOHAMCD Ct aL, 1964) . These results show that the crude venom of Bitis arietans venom is heterogenous in nature and consists of a number of components, each of which is responsible for one or more of the pharmacological properties of the venom. For example, when equal concentrations were used, fraction 1 was the most powerful depressant of the heart, fraction 2 was the most potent in increasing capillary permeability and in producing hypotension, while fraction 3 was the most powerful inhibitor of the rabbit intestine and in antagonizing acetylcholine induced contractions on smooth muscles. TOXJCONl971 Yol. J2
glut arletons Veaom
375
Ackeowla¢ement-We are gratdul to Mr. Mit L Et.-Ha~ o for valuabk technical assistance aced Mr. A. EtrTaur for photographic assistance. REFERENCES Baoent .Y, D. G. (1968) The venomous snaky of Central and South Africa. In : Ysnonrous Anirrn/s and their Vertoms, p. 403, (Bvc~tu.. W., Bvcu~v, E. and Deur oFev, V., Eds.) . New Yorlt: Academic Pros . Bürs+auva, E. (1946) Observations on the iwlated phrenic nerve diaphragm preparation of the rat. Br. J. Pharnme. Chemother. 1, 38. C~wrwarr, D. S. (1968) The symptomatology, pathology and treatment of venomous snakes of Central and Southern Africa . In : Yenonaus Animals and theb Yenonrs, p. 463, (Bvct~rtt, W., BUCICIFY, E. and Devto~u, V., Eds.). New York : Academic Pros . Croeareraerr, P. A. (1968) The venorns of Central and South African snakes. In : Yenonaus Anlnah and their Yerams, p. 437, (BuctrEat, W., HUC[IBY, E. and Devt.ot+eeu, V., Eds.) . New York : Academic Pass. DFS.rrewu<, G. R. (1968) Studies on African snake venoms-11. Differentiation between Proteinase and amino acid esterase activities of some African viperidae venoms. Toxlcon 6, 103. Fnvrcew~rr, B. (1930) On the naturt of inhibition in the intestine. J. PhysloL, Loral. 70, 145. Fasvvoaec, T. A. (1%S) Quaatity and toxicity of Bltis Jaclusis venom. Acts trop. 22, 267. Grtna, S., Awer., S., GQ.AT, G., Soivtvtrw, T. and WEGWART, Y. (l%3) Neutron activation analyses of snake vcnoms : presence of copper. Nature. Loral. 197, 383. GR~sssr, E., Baecxsvet.eR, T., Scrtw~az, D. E. and Potvaa~n, E. (1956) Comparative analysis and ekrtrophoretic frtctionations of snake venoms. In : Yerroms. Pub. No. 44, Amer. Assoc. Adv. Sci., Wash., D.C., p. 153. Issr~It., M., OSMAN, O. H., Isaxrrt~, S. A. and E~-ASMAR, M. F. (1972) Cardiovascular and respiratory response to the venom from the scorpion Leiurus quinquestriarus. E. Afr, med. J. 49, 273. De J~t.otv, P. G., BAY BAYO, J. and De JALO~I, M. G. (1943) Farmacoterap . Actual3, 313. In : Pharmacological Experiments on Isolated Preparotlons (1968), p. 92, (The Staffof Pharmacology Dept ., Univ. of Edinburgh, Eds.). Edinburgh: E. & S. Livingstonc. JUDAH, J. D. and Wtts.ovarrav, D. A. (1%2) Quantitative method for the study of ppillary pernteability : extraction and dctcrmination of trypan blue in tissues . J. path . Bact . 83, 567. Mactvus, R. (1904) PJlugers Arch. ges . Physlol. 102, 123. In : Pharrrwcologlrnl Experiments on Isolated Preparations, (I96ß), p . 62, (The Staff of Pharmacology Dept ., Univ. of Edinburgh, Eds.). Edinburgh : E. do S. Livingstonc. MARKS, V. (1959) An improved glucose oxidase method for determining blood, ccrcbrospinal fluid, and urine glucose kwels. Cllnlca chim . Acts 4, 395. MUIIAMFA, A. F1 ., Ecseaouur, M. and KAINt:L, A. (1964) Effects of Walterinessia aegypta venom on blood sodium, potassium and catxholamina, and urine 17-kctosteroids . Toxicon 2, 103. OSMAN, O. H., Isst~u., M. and Et.-Asatiea, M. F. (1973) Pharmacological studio of snake (Dendroaspit angusticeps) venom. Toxirnx 11, 185 . Taus, A. M. and Eu.rorr, W. B. (1964) I-Some effects of snake venom on mitochondria . Toxkon 2, 87 . Torahs., A. (l%S) Tire cffoct of four monoaminc oxidase inhibitors on the rat uterus. Br. J. Plrarmac. 25, 217. V~CIt, J. A., G~ucrrr~, H. P. and M~rrreel, J. H. (1967) Pathophysiological studies of ten snake venoms . In : Anima) Toxins, p. 269, (Russets., F. E. and S~uNOeas, P. R., Eds.) . Oxford : Pergamon Press. Zatu, O. A., Kttoaats, A., Asnet.-R~urst~ry Y. and Et.-Naanr, S. (1970) Circulatory shock caused bY a toxic fraction from EchLt earlnatus venom. Ain Shams med. J. 2l, 647.
TOXICON 1971 Vot. 11
Yot.
17, Op. 169-373. t~apmoo Pra Teino~d la üert Drhaie .
PHARMACOLOGICAL STUDIES OF SNAKE (B1TIS ARIETANS) VENOM O. H. 05C+uIV and K. A. GuM~A Departments of Pharmacology and Biochemistry, University of Khartoum, Sudan (~lccepred for publication 23 btay 1974) O. H. Osr4rty and K. A. Gtn~tM . Pharmacological studies of snake (Bins arlerans) venom. Toxkwr 12, 369-373, 1974 .-Venoun from Bins arierans was fractionated on Sephadea G-100 into flue main fractions, of which only fraction 1 was lethal to mice . The crude venous and fractions 1 and 2 produced a gradual and precipitous fall in the blood pressure of the cat . This fall was not antagonised by prctrsatment with hexamethonium, cyproheptadine, propranobl, or atropine . Also, it was not blocked by indomethacin or the kinin inhibitor, trasylol . Both the crude venom and fraction 1 produced a depression of the isolated bean which was not prevented by atropine or hexamethonium. Similarly, the crude venom and fractions 1 and 2 increased the capillary permeability in rabbits. Thecrude venom and fractions 1 and 3 pradrrced an inhibition of a variety of smooth muscle preparations such as the rabbit intestine, guineapig ileum and the rat uterus . However, the spontaneously contracting rat uterus was stimulated by the crude venom and fraction I. The venom produced hyperglycaemia in the rabbit and a decrease in the potassium level of the cat blood. It is suggested that the circulatory shock produced by the venom is due to a direct inhibition of vascular smooth muscle, together with a direct dcpr+casion of the myocardium and an increased capillary permeability . INTRODUCTION
Bltls arletans, the puff' adder, is one of the commonest and most widespread of African snakes (BROADLY, 1968). It has been claimed to be responsible for most cases of snake bite in Central and Southern Africa (C1IAPMAN, 1968) . Its venom was said to be primarily cytotoxic (CHAeMAN,1968), and to produce both local and genernlized internal haemorrhagic lesions (CHRt3TFNSEN, 1968). GnASSE~r et al. (1956) had fractionated the venom into 4 or 5 components by the use of paper electrophoresis, all of them migrating towards the anode. The crude venom consists of a complex mixture of proteinases and amino acid esterases (DELPIERRE, 1968) . Gtrrtat et al. (1963) found that the venom also contained copper. The Ln~ of the crude venom was determined by Fttt:YVOCt=.I. (1965) in mice to be 1-2 ltg per g when injected subcutaneously . The main pharmacological effects of the crude venom are hypotension, and bradycardia (Vtctc et al., 1967), collagenolytic activity, anticoagulant activity (Ctttt.tsTExserr, 1968), and inhibition of mitochondria) respiration (TAUB and Er.uorr, 1964) . The present study was carried out with the aim of examining in some detail the pharmacological properties of the crude venom and some of its fractions on different isolated tissues and animal preparations. MATERIALS AND METHODS
Lyophilized Bias arietans venom was obtained from Sigma Chemical Company, St. Louis, Missouri, U.S.A. Fractionation of the crude venom was performed using Sephadex G-100 (Sigma) column with bed dimension of2~5 x 35 cm equilibrated with 001 M phosphate buffer, pH 7~4. Twenty milligrams of dried venom dissolved in 3 ml of phosphate buffer roxrcox t9m r o r. u
369
370
O. EL 06MAN and IL ti GIJMM
wtre applied to the column. Flution was continued with 001 M phosphate buffer and 5 ml fractions were collected at flow rates of 35-40 ml per hr. Protein was estimated spectrophotometrically at 280 nm. Solutions of the crude venom or the fractions wen kept at 4°C until used. Blood pressure recording was carried out in cats (IseutL et al., 1972). The isolated heart (Langendorfl) preparation was performed using rabbits (Os~N et al., 1973). The assessment of vascular permeability was carried out by an adaptation of the method of Jtrn~t and WILLOUGHHY (1962) . The abdominal skin of adult albino rabbits was shaved and 24 hr later the animals were injected i.v. with Evans blue dye (7 mg per kg). The crude venom or the fractions were injected i.d. on both sides of the midline of the shaved area. Twenty minutes later, the estimations of the dye leakage were made by the macroscopic appearance of the skin . Blood glucose was determined according to the method of MARKS (1959). Rabbits of either sex weighing between 1"5 and 2 kg were fasted overnight. The venom was injected i.v. in a dose of 300 ~g per kg. Blood samples were withdrawn from a marginal ear vein before and at 30 min intervals aRer injection of the venom for a periôd of 2 hr. Sodium and potassium were determined in cats using whole blood rather than serum in order to avoid errors that might arise as a result of the haemolytic effect of the venom. Both sodium and potassium were determined by the flame photometer method . Cats of either sex weighing between 2 and 4 kg were anaesthetized with pentobarbitone sodium (40 mg per kg i.p.). The venom was injected i.v. in a dose of 300 ~g per kg through a femoral vein. Blood samples were taken from a common carotid artery before and at } hr intervals over a period of 3 hr after injection of the venom. The venom and its fractions were tested on the following preparations : the isolated rabbit intestine (MAGNUS, 1904), guinea-pig ileum, the isolated pcriartcrially stimulated rabbit jcjenum (FINKGLMAN, 1930), the isolated rat uterus (Dt: JALOIY et al., 1945), the spontaneously contracting rat uterus in Ringer-Locke solution ('To'rt11LL, 1965), and the isolated phrenic ncrvo-hcmidiaphragm of the rat (BGLaRtHa, 1946). The following drugs were used : acetylcholine chloride (Roche), atropine sulphate (The British Drug Houses Ltd.), cyproheptadine (Merck Sharp & Dohme), hcxamcthonium chloride (Fluka), histamine acid phosphate (British Drug Houses), indomethacin (Merck Sharp & Dohme), phenoxybenzamine hydrochloride (Smith Kline & French), propranolol (LC.L), serotonin creatinc sulphate (British Drug Houses), and trasylol (5000 kallikrein inactivation units per ml, Bayer). 1tFSULTS
Fractionation of the crude venom of Billsarietans by Sephadex G-100 revealed 5 fractions (Fig. 1). Only fraction 1 was lethal to mice when injected in a dose of 1 ~g per g i.p. Shortly after injection there was motorexcitement and restlessness which was followed by depression and finally convulsion and death 1-2 hr later. Autopsy revealed haemorrhage in the lungs and kidneys. The other four fractions were non-toxic when given in doses between 05-1 ~g per g i.p. No peculiar behaviour or symptoms were observed . Eß"ect ojtbe
venom on
arterial blood pressure (Fig. 2)
Intravenous injection of the crude venom in doses of 50-300 ~g per kg produced after a latent period of 10-20 sec a gradual and marked fall in the blood pressure of anaesthetized cats that persisted for 30 min or more and then returned to near-normal levels . Repeating TOXICON I971 Vd. l1
Bi~ir arietana Venom
,7i .
571
..
~n
1
;n -
t
I `ubR na
~u
f~~
I~" "ti ~. sv nv
(eaGh S ml
FI(F . I . FRACTIONATION OF 2O mti B. aI1C1!!!!S VF;NOM ON SEPIIADEX G~I00 COLUMN ; O'OI PF105PHATE SUFFCR, pFl 7-4 . vras 280 nm .
Protein
M
cstimatcd spoctrophutumctrically at
the injection of the venom caused a further fall in blood pressure that reached a very low level and resulted in circulatory shock :tnd death of the animals, Both fr :tctian I and 2 produced hypotension when injected in doses of 25-SU Ng per kg. however, when cyual conccntrttions were used, fraction 2 was more potent than either fraction I or the crude venom. Other fractions did not produce any significant etï'~~a on the arterial blood pressure . The hypotensive elfcct of the venom or its fractions were not antagonized by pretreatment with Itexamcthottium (7 mg peT kg), cyproheptadine (2 mg pc;r kg), propranolol (0-5 mg per kg), atropine (2 mg per kg), indomethacin (25 mg per kg), or trasylul (l ml per kg) . The venom produced a degrease in the rate and depth of respiration which was transient and returned to normal despite the fall in blood pressure. Usually arrest of respiration was preceded by circulatory arrest . E,Eject of the venom on the isolated rabbit heart ( Fig . 3) The crude venom in doses of 50-100 pg produced a marked decrease in both the force and rate of cardiac contraction . This cardiac depressing activity was not blocked by atropine (3001tg), or hexamethonium (l mg) . Fraction l also produced a very conspicuous depressant elTect on the heart but fraction 2 was less elFective in this respect. The other fractions were without activity. Effect of the venom on capillary permeability The crude venom in doses of 50-100 ~g per kg produced an increase in capillary permcabi(ity in the rabbit as assessed by the degree of dye leakage into the skin 20 min after injection of the venom. The average diameter of the blue area (Mean f S.E.) obtained from TOXICON 197 : Yal. ! :
372
O. H. 05MAN and K. A. GUMAA
mmHg 140
loo so Zo
mmHg loo so
r~
20
. (d)
. (e)
"
(f)
" ~(g)
FIU. 2. TIIE LFFFt"r of ~. Qr1l'i(III.K VF~~t on 7H1: CAr OLOt1P PRF:StiURE,
'The vcltunt was injected at A, li, C, U, t:, F and G (IW lag per kgl. Flcxmethonium, 7 mg per kg was injected 70 min txfurc li . Cypn~hcptadine, 2 mg per kg was injected 10 min lx:fun: C . Propranulol, 0~5 mg per kg was injected l0 min before f) . Atropinr, 2 mg per kg was injatcd 10 min before E. Indultxxthacin, 25 mg per kg was injected i.p . 30 min before F . Trnsylul, l ml per kg was injected 20 min before G .
the vcnum-treated animals was 24 ~: l'I mm as compared to 4 -~ 007 mm obtained from the saline-treated controls. Six rabbits were used in each test, Extensive oedema was still present at the site of injection several days after the venom injection . Similarly, both fractions l and 2 produced extensive capillary permeability when injected in doses of 20 pg per kg. Fraction 2 was much more potent in this respt:ct. The average diameter of the bloc area in mm (Mean ~ S.E.) obtained from 4 animals each for fractions 1 and 2 were 27 f 2'0 mm and 31 ~ l'S mm, respectively. The capillary permeability produced by the venom or the fractions was not modified by pretreatment with either cyprohcptadine (1 mg per kg), trasylol (1 mg per kg) or indomethacin (25 mg per kg). E.(ject of !!te t"enom on isolalel! smo~~th musc% preparaJiorrs The pendular movement of the isolated rabbit intestine was significantly inhibited by the crude venom (5-10 )tg per ml) . Only fraction 3 caused an inhibition of the pendular movement of tht: intestine in doses of 1-2 pg per ml. Other fractions were inclFcctivc or produced slight inhibition (Fig. 4). This inhibitory elTcct was not blocked by phenoxybenzamine (1 Itg per ml) and/or propranolol (l Ng per tnl) . TOXICO~V 1971 F'ul. !:
min
(a)
(b)
(c)
(d)
(e)
4 r
cm (a)
(b)
(c)
(d)
(e)
(f)
3 . ~Î111 : I:FFFCy u( />i. arirluus VENUh1 ()N THE IS()LATF.U RAHHITS HEART . The venom w ;as injecteJ at (a), (hl anJ (c) in a J(rc uf SU Ftg pcr hcart . At (J), fraction I was injmtml (25 lagl . At (ch fra(aion 3 was injcctcJ ('_5 lagl . Atropine, 3(1(1 lag was injectcJ IU min Ixfurc (hl . Hexantethuniunt, t mg wa+ injmtcJ 10 min befurc (c) . F~Ir : .
hIC: . -( . TI1l. rFFE(T OF
R. urirlune
VF:NIIM uN '111L PLNUULAR MUVEMF.Nr OF TIIE RAültfr~ti 1 N~rF 3 rl NE .
At (al, R. r)rirrmr venom IU lag per ml was aJJeJ tu the bath . At (h), fraction I (5 Elg per n)1) ; Ic), fr;tction Z (5 lag per ml) ; (J1, frac(iult 3, (3 lag per ntll : (el. fraction 4, (5 !lg per mll anJ If 1, fraction S, (S lag per ntl> were aJJcJ . Bath vohlnte, 25 ml .
ro .rico.v ry-.r t-.,1 .
1:
r.,,.
sr_
r
crn
F'II, . $. I~III~ F11~1('~1 (+I I1. ((r7l'1(lll.+' Y'l .!~(HI (+\ IIII . tiIKIN~f "1til~++l1":LY' ('(IVIKAI'l'Itrli KA~r 111~I~Kl ;ti. Ital utrru, in ocstrua, hath culunx: III ntl, tcntl>Lralurc 37 C-.At (a), R.urirtune acnom,2(lug Ixr ntl, vca+ aililcJ to thc hath . At (.h), fraction I (10Icg Ixr ntl) aml at fcl, fraction ' (10 y,g Ixr ml) wcrc ad~lcd .
lm .rrcm.v 14v Iv,l. 1_
BItU nrietanr Veaom
373
Both acetylcholine (0~2 ltg per ml) and histamine (0~1 Irg per ml) induced contractions on the guinea-pig ileum preparation which were inhibited molt: than 80 per ant by concentrations of the crude venom ranging between 5 and 10 ~g per ml. Similar results were obtained on the rat uteruu preparation in de Jalon solution when 5-hydroxytryptamine (0" 1 Ng per ml) was used as the agonist. Fraction 1 (5 pg per ml) completely antagonized histamine contractions on the guinea-pig ileum but it had little activity against acetylcholine induced contractions, while fraction 3 mainly antagonized atxtylcholine . Fractions 2, 4 and 5 were inactive. The crude venom (20 Itg per ml) and fraction 1 (10 ~g per ml) produced a stimulant activity on the spontaneously contracting rat uterus in Ringer-Locke solution ; the frequency and amplitude of contraction being increased (Fig. S). Fraction 2 produced a slight effect, while other fractions had no effect on this preparation . Neither the crude venom, nor any of its fractions had any effect on adrenergic transmission as evident from their effect on the periarterially stimulated rabbit jejenum. Also, they had no action on neuromuscular transmission as evident from their effect on the isolated phrenic nerve-hemidiaphragm ofthe rat . EJfec~ of the venom on blood glucose, sodium and potassirun Intravenous injection of the crude venom (300 Itg per kg) into fasting rabbits produced a significant rise in blood glucose level which reached its maximum 1 hr after injection and then began to return to normal level by the end of a 2 hr period . Similarly, when the venom was injected i.v . (300 4tg per kg) in anaesthetized cats, it produced a significant decrease in potassium level of the whole blood that reached its maximum 2 hr after injection and gradually cost again to prcinjcction Icvcl after a 3 hr period . Sodium levels however, were not significantly alTected (Table 1). TASLe l . EFFecr OF
Gluoose (mg/100 mp Na* (mequiv./I) K" (mequiv./l)
0 93 t 4ß (6)' 144 t 6~3 (3) 40~3 t 1~1 (3)
Buts artaanr veNOw, (300 ~g per kg i.v.) IN RAaaR3 AND Na+ AND K* LëVE[-t IN 30 128t f 6~4 (ti) 143 f 6~2 (3) 30t f 32 (3)
Blood levels aRer Injection 60 90 120 I32t 133t 109t t t t 6~2 4~2 3~2 (t7 (6) (6) 143 140 I4l t f f 3~1 8~9 10~6 (3) (3) (3) 26~6t 23~6t 23~8t f f f 1-0 1~3 0~33 (3) (3) (3)
ON BLOOD OLUCOÔE LEVEL CATS
130 100 t 3~9 (6) 142 f 11~6 (3) 32~5 f 3~8 (3)
" lagures in parentheses indicate the number of animals used. Results arc presented as means f standard error. fi Values are :ignificantly different from the oontmls (P<0-03). DISCUSSION
180 98 f 4~3 (6) 142 t 11~6 (3) 33 f 1~7 (3)
(min)
The principal clinical effects of envenomation by viper venom are local and generalized internal haemorrhagic lesions which may be so severe as to cause immediate death (CIIRISTGNSEN, 1968). The present studies show that Bibs arfetans venom could be fractionroxicoN t9~~ vor. r:
S74
O. H . 05MAN and K. A. GLT1VtM
ated on Sephadex G-100 into 5 main fractions. This is in agreement with the results of Ga~~ et al. (1956), who used paper electrophoresis. Only fraction 1 was lethal when injected into mice, and produced haemorrhagic lesions in both the lungs and kidneys. It is evident from the experimental results that at least three factors arecontributing to the circulatory shock caused by the venom or its fractions. Firstly, a direct inhibitory effect of the vascular smooth muscles resulting in vasodilatation of the arterioles. This effect was notantagonized by ganglionic blocking agents, antihistaminic, antiserotonergic, ß-adrenergic receptor blocking drugs or cholinergic blockers. Similarly, it was not counteracted by drugs which inhibit prostaglandin or kinin actions such as indomethacin and trasylol . Secondly, we observed a direct depressant action on the myocardium, and thirdly, an extensive increase in vascular permeability that was not modified by pretreatment with either antihistamines, antiserotonergics, or agents which inhibit kinins or prostaglandins . Vtctc et al. (1967), using dogs, showed that evisceration did not prevent the hypotensive effect of Bibs arietans venom but eliminated the bradycardia. They suggested that the venom did not produce a marked pooling of blood in the hepato-splanchnic bed ; however, pulmonary pooling might be responsible for the hypotension. It has been shown by 7.~xt et al. (1970) that the increase in capillary permeability produced by Echis carinatus venom or one of its fractions was prevented by pretreatment with the antihistaminic drug promethazine . In the present studies, however, promethazine was ineffective in antagonizing either the hypotension or the increase in capillary permeability produced by Bitis arietans venom or its fractions (unpublished observations). It is known that Bitis arierans venom contains a complex mixture of proteinases and amino acid esterases (Decr~exRe,19G8), and these enzymes may produce some breakdown products in the body such as adenosine, lysolecithin, certain fatty acids, and histamine which may have deleterious clTe~cts on the heart and circulation (CFIRISTIiNSCN, 1968). The effect of the crude venom and some of its fractions on smooth muscles scans to be generally inhibitory in nature and directly mediated. The spontaneously contracting rat uterus preparation is a notable exception, since both the crude venom and fraction L increased the frequency and amplitude of uterine contraction . This stimulant effect was not antagonized by either atropine, hcxamcthonium or mcthysergide but was partially blocked by indomethacin indicating that the effect may be mediated through release of prostaglandins and/or kinins (unpublished observations). Neither the venom nor any of its fractions affected adrenergic transmission or neuromuscular transmission of skeletal muscles. The hyperglycaemic effect of the venom may be due to a stress reaction resulting in the liberation of catecholamines from the adrenal medulla; however, this needs confirmation . Potassium levels of the whole blood were decreased by about 41 per cent 2 hr after injection of a sublethal dose of the venom. Sodium levels, however, were not significantly changed. Potassium lowering may be due to the increased release of ACTH from the anterior pituitary gland (MOHAMCD Ct aL, 1964) . These results show that the crude venom of Bitis arietans venom is heterogenous in nature and consists of a number of components, each of which is responsible for one or more of the pharmacological properties of the venom. For example, when equal concentrations were used, fraction 1 was the most powerful depressant of the heart, fraction 2 was the most potent in increasing capillary permeability and in producing hypotension, while fraction 3 was the most powerful inhibitor of the rabbit intestine and in antagonizing acetylcholine induced contractions on smooth muscles. TOXJCONl971 Yol. J2
glut arletons Veaom
375
Ackeowla¢ement-We are gratdul to Mr. Mit L Et.-Ha~ o for valuabk technical assistance aced Mr. A. EtrTaur for photographic assistance. REFERENCES Baoent .Y, D. G. (1968) The venomous snaky of Central and South Africa. In : Ysnonrous Anirrn/s and their Vertoms, p. 403, (Bvc~tu.. W., Bvcu~v, E. and Deur oFev, V., Eds.) . New Yorlt: Academic Pros . Bürs+auva, E. (1946) Observations on the iwlated phrenic nerve diaphragm preparation of the rat. Br. J. Pharnme. Chemother. 1, 38. C~wrwarr, D. S. (1968) The symptomatology, pathology and treatment of venomous snakes of Central and Southern Africa . In : Yenonaus Animals and theb Yenonrs, p. 463, (Bvct~rtt, W., BUCICIFY, E. and Devto~u, V., Eds.). New York : Academic Pros . Croeareraerr, P. A. (1968) The venorns of Central and South African snakes. In : Yenonaus Anlnah and their Yerams, p. 437, (BuctrEat, W., HUC[IBY, E. and Devt.ot+eeu, V., Eds.) . New York : Academic Pass. DFS.rrewu<, G. R. (1968) Studies on African snake venoms-11. Differentiation between Proteinase and amino acid esterase activities of some African viperidae venoms. Toxlcon 6, 103. Fnvrcew~rr, B. (1930) On the naturt of inhibition in the intestine. J. PhysloL, Loral. 70, 145. Fasvvoaec, T. A. (1%S) Quaatity and toxicity of Bltis Jaclusis venom. Acts trop. 22, 267. Grtna, S., Awer., S., GQ.AT, G., Soivtvtrw, T. and WEGWART, Y. (l%3) Neutron activation analyses of snake vcnoms : presence of copper. Nature. Loral. 197, 383. GR~sssr, E., Baecxsvet.eR, T., Scrtw~az, D. E. and Potvaa~n, E. (1956) Comparative analysis and ekrtrophoretic frtctionations of snake venoms. In : Yerroms. Pub. No. 44, Amer. Assoc. Adv. Sci., Wash., D.C., p. 153. Issr~It., M., OSMAN, O. H., Isaxrrt~, S. A. and E~-ASMAR, M. F. (1972) Cardiovascular and respiratory response to the venom from the scorpion Leiurus quinquestriarus. E. Afr, med. J. 49, 273. De J~t.otv, P. G., BAY BAYO, J. and De JALO~I, M. G. (1943) Farmacoterap . Actual3, 313. In : Pharmacological Experiments on Isolated Preparotlons (1968), p. 92, (The Staffof Pharmacology Dept ., Univ. of Edinburgh, Eds.). Edinburgh: E. & S. Livingstonc. JUDAH, J. D. and Wtts.ovarrav, D. A. (1%2) Quantitative method for the study of ppillary pernteability : extraction and dctcrmination of trypan blue in tissues . J. path . Bact . 83, 567. Mactvus, R. (1904) PJlugers Arch. ges . Physlol. 102, 123. In : Pharrrwcologlrnl Experiments on Isolated Preparations, (I96ß), p . 62, (The Staff of Pharmacology Dept ., Univ. of Edinburgh, Eds.). Edinburgh : E. do S. Livingstonc. MARKS, V. (1959) An improved glucose oxidase method for determining blood, ccrcbrospinal fluid, and urine glucose kwels. Cllnlca chim . Acts 4, 395. MUIIAMFA, A. F1 ., Ecseaouur, M. and KAINt:L, A. (1964) Effects of Walterinessia aegypta venom on blood sodium, potassium and catxholamina, and urine 17-kctosteroids . Toxicon 2, 103. OSMAN, O. H., Isst~u., M. and Et.-Asatiea, M. F. (1973) Pharmacological studio of snake (Dendroaspit angusticeps) venom. Toxirnx 11, 185 . Taus, A. M. and Eu.rorr, W. B. (1964) I-Some effects of snake venom on mitochondria . Toxkon 2, 87 . Torahs., A. (l%S) Tire cffoct of four monoaminc oxidase inhibitors on the rat uterus. Br. J. Plrarmac. 25, 217. V~CIt, J. A., G~ucrrr~, H. P. and M~rrreel, J. H. (1967) Pathophysiological studies of ten snake venoms . In : Anima) Toxins, p. 269, (Russets., F. E. and S~uNOeas, P. R., Eds.) . Oxford : Pergamon Press. Zatu, O. A., Kttoaats, A., Asnet.-R~urst~ry Y. and Et.-Naanr, S. (1970) Circulatory shock caused bY a toxic fraction from EchLt earlnatus venom. Ain Shams med. J. 2l, 647.
TOXICON 1971 Vot. 11