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CAMPHELL, C. H. (1967) The Taipan (Oxyuranus scuteltatus) and the effects of its bite . Med. J. Aust. 1, 735 . COVACEVICFI, J . and Wonmsr, J. (1976) Recognition of Parademansia mlcrolepldotus McCoy (Elapidae), a dangerous Australian snake . Proc. R. Soc. Qd 87, 29 . Deoltas, P . J . (1971) The story of some Indian poisonous snakes . ln : Venomous Animals and Their Yenoms, (BUI~RL, W, and BUCKLEY, E. E ., Eds .) . New York : Academic Press . FAiRLEY, N. H . and SPLa~1-r, B. (1929) Venom yields in Australian poisonous snakes. Med. J. Aust. 1, 336 . FnvrreY, D . J . (1964) Statlstlcal Method in Biological Assay, 2nd Edn., p . 524. London : Griffin. Fae>:nulv, M . and K$LlawnY, C . H . (1934) The venom yields of common Australian poisonous snakes in captivity. Med. J. Aust. 2, 190 . GAMHAVORN, S . (1969) Toxicities of Thailand snake venoms and neutralization capacity of antivenene . Toxlcon 7, 239. KELLAWAY, C. H . (1931) Observations on the certainly lethal dose of venom of the common brown snake (Demansta textilis) for the common laboratory animals . Med. J. Aust . 2, 747. KFL1.nwnv, C . H . and THOMSON, D . F . (1932) Observations on the venom of a melanotic insular variety of the tiger snake (Notechis scutatus). Aust . J. exp. Biol. med. Scl . 19, 35 . Mnvzox, S. A . and MlrrroN, M . J . (1969) Properties and composition of venoms . In : Venomous Reptiles, p . 34. London : George Allen & Unwin Ltd . Mulmrr, S . A . (1974) Snakes and snake venoms . In : Venom Diseases . Springfield : Charles C. Thomas . RussEC.L, F . E . (1966) To be, or not to be . . . the Ln, e . Toxlcon 4, 81 . SUTF1E1tLAND, S . K., BROAD, A . J., TANNER, C . and COVACEVICH, J . (1978) Australia's potentially most venomous snake : Parademansta microlepidotus. Med. J. Aust. i, 288 . WORRELL ., E . (1970) Reptiles ojAustralla . Sydney : Anges 8c Robertson .

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Toxfcor . Vol. 17, pp . 664-667. Peraamon Press Ltd. 1979 . Printed in Groat Hritaln.

m

DISRUPTION OF ELECTROCARDIOGRAPHIC ACTIVITY BY STREPTOLYSIN O IN RATS Susr~n GUPTA

and

R. K.

GuPTA

Departments of Physiology and Pathology, Maharani Laxmi Bai Medical College, Jhansi-284001, India (Accepted for publication 29 May 1979) RHEUMATIC fever is a post-streptococcal sequel whose pathogenesis is not well known. Streptolysin O, a potent streptococcal exotoxin produces cardiac damage (GUPTA et al., 1973 ; GUPTA et al., 1976a ; HALSERT et al., 1961) and derangement of cardiac activity in experimental animals (GUPTA et al., 1976b, 1976c, 19764; GUPTA and GUPTA, 1977; SPIRA et al., 1968). Streptolysin O has also been shown to be toxic to mammalian heart cells in tissue culture (TrIOMPSON et al., 1970) . In the present study the effect of streptolysin O on the electrocardiogram (ECG) of rats has been investigated. The experiments were performed on 52 Swiss albino rats of either sex weighing 150 ~ 20g and anaesthetized with i .p . pentobarbitone sodium (35 mg/kg) . Half an hour after induction ~f anaesthesia electrocardiograms were recorded using needle electrodes . The effect

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of streptolysin O (2, 5, 10 and 20 haemolytic units/kg, in 0~5 ml ; one haemolytic unit is equivalent to 1 minimum haemolytic dose at the 50~ haemolytis end point) injected i.p. at 30 min intervals, on the ECG was studied in untreated rats and also in those pretreated i.p. (30 min prior) with (1) atropine sulphate (Labros Chemicals, Lucknow, India) in doses of 1 and 2 mg/kg ; (2) mepyramine (May & Baker Ltd., Bombay, India) in doses of 25 and 50 mg/kg; (3) dexamethasone (Merk Sharp & Dohme, Bombay, India) in doses of 0~2 and 0~5 mg/kg ; (4) antistreptolysin O (Wellcome Reagents Ltd., England) in doses of 10 and 20 i.u./kg. Animals similarly receiving streptolysin O inactivated by prior treatment with varying doses of cholesterol, antistreptolysin O (37°C for 30 min) or heat (60°C for 30 min) served as control. Four animals were used for each test . Animals were maintained under standard conditions and were not reused earlier than 2 weeks. Partially purified, reduced and lyophilized streptolysin O from group A (Human Institute of serobacteriological production and research, Budapest, Hungary) and group C haemolytic streptococci and lyophilized antistreptolysin O (Wellcome Reagents Ltd., England) were reconstituted in sterile isotonic phosphate buffered saline (pH 7~2) just before use. The streptolysin O preparations from group A and group C streptococci contained 2000 and 3500 haemolytic units/mg protein respectively and were in the reversibly oxidised state (approximately 96 ~). Polyacrylamide disc gel electrophoresis revealed a major protein band (streptolysin O, 92 ~) followed by a second minor band (8 ~). Antistreptolysin O revealed a single precipitin band against streptolysin O in immune precipitation electrophoresis. The cholesterol suspension was prepared by the method of CoxEN et al. (1937) ;01 ltg of cholesterol completelyinhibited one haemolytic unit of streptolysin O. Administration (i.p.) of streptolysin O in doses of 2-5 haemolytic units/kg did not produce any obvious effect on the ECG in rats, however, in 75 ~ of the animals a dose of 10 haemolytic units streptolysin Oper kg produced within 60-90 sec a reduction of 15 ~ 2~6 in the heart rate ; this effect was transient and completely reversible, lasting only 2-3 min after the injection . In higher doses (20 units/kg) it produced progressively increasing bradycardia and derangements of atrioventricular and intraventricular conduction processes . Elevation of the ST segment was observed as a preterminal event in 50 ~ of these animals; all electrical activity of the heart ceased within 5-7 min after the injection. These changes are shown in Fig. 1 . Streptolysin O, both from group A and group C haemolytic streptococci, produced almost identical ECG changes. Rats receiving streptolysin O inactivated by cholesterol, antistreptolysin O or heat did not show any ECG changes. Administration (i .p.) of atropine sulphate, mepyramine, dexamethasone or antistreptolysin O per se did not produce any ECG alterations in rats . ECG changes produced by challenging doses of streptolysin O (20 haemolytic units/kg, i .p.) in rats pretreated with atropine sulphate or mepyramine were similar to those seen in untreated animals receiving active streptolysin O. On the other hand similar doses of streptolysin O failed to evoke any ECG changes in rats pretreated with dexamethasone or antistreptolysin O. Administration (i.p.) of streptolysin O (20 haemolytic units/kg) produced progressively increasing impairment of intraca.rdiac conduction processes terminating in cardiac standstill within ~7 min after injection. Similar ECG changes have also been observed after i.v. administration of streptolysin O in other mammals (Gurrn and GurTa, 1977 ; HALBERT et al., 1961 ; HALPERN and REHMAN, 1968). These observations are also consistent with the demonstration of a myocardial depressent effect of streptolysin O on the isolated and intact mammalian and amphibian heart (Guprn et al., 1976b, I976c, 1976d; HALPERN and REHMAN, 1968 ; KELLNER et al., 1956 ; RE1TZ et al., 1968). Since streptolysin O inactivated with cholesterol, antistreptolysin O or heat (agents which inhibit this toxin, THOIVIPSON

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No ECG changes were seen in animals receiving streptolysin O inactivated by heat, cholesterol or antistreptolysin O. Challenging doses of streptolysin O (20 haemolytic unitslkg, i.p .) did not produce any ECG changes in rats pretreated with deaamethasone or antistreptolysin O, however, the ECG changes by streptolysin O in animals pretreated with atropine sulphate or mepyramine were similar to those observed in untreated rats. The P-R interval was 004 and 0"06 aec at 0 and 60 sec respectively . The ST segment was isoelectric at 0 and 60 sac and elevated at 150, 180 and 300 sec. The Q-T inteval was 0"06 sec at 0 and 60 sec and 008 sec at the other times.

et al., 1970) failed to produce any changes in the ECG, cardiotoxicity as observed in animals receiving active streptolysin O was probably due to this toxin and not due to any other streptococcal component contaminating streptolysin O. These observations suggest that streptolysin O is cardiotoxic, however, the mechanism is not well known. RErrZ et al. (1968) have suggested that streptolysin O produces this effect by the release of acetylcholine . Since pretreatment of animals with atropine sulphate failed to protect them against the cardiotoxic effects of streptolysin O, these changes do not seem to be due to cholinergic stimulation . Failure of atropine to block the myocardial depressant effect of streptolysin O on cat and frog heart in situ (GuprA et al., 197Cx~ also supports this suggestion. Failure of mepyramine (an antihistaminic drug) pretreatment to protect the rats against the cardiotoxic effects of streptolysin O would suggest that these effects of streptolysin O are not mediated via the release of histamine . The rapidity of onset

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of cardiotoxic effects as observed in the present as well as preceding studies (GUPTA et al., 19766, 1976c, 19764; GurrA and GurrA, 1977 ; HALPatx and R$HhIAN, 1968 ; KELLNER et al., 1956 ; Rerrz et al., 1968) suggests that streptolysin O may have a direct toxic effect on the myocardium and intracardiac conduction processes. The observations ofTxolutrsox et al. (1970) that streptolysin O is capable of rapidly permeating into the mammalian heart cells in tissue cultures are consistent with this suggestion. Protection against the cardiotoxic effects of streptolysin O by pretreatment of rats with dexamethasone, a substance known to stabilize the cell and lysosomal membranes (Si3YSRS and TRAVIS, 1971), is also consistent with this suggestion . Acknowledgements-We are grateful to the Indian Council of Medical Research, New Delhi, India, for financial support. REFERENCES Coxex, B., SCHWACHMAN, H, and PErtKnvs, M. E. (1937) Inactivation of pneumococcal haemolysin by certain sterols. Proc. Soc. exp. Biol. Med. 36, 586. Gurra, S. and Gurra, R. K. (1977) Studies on toxic effects of streptolysin O: electrocardiographic changes in mammals. Indian J. Physiol. Pliarm . 21, 199. Game, R. K., Mr~oriu, R. M. L. and CH~ruRVSnt, U. C. (1973) Cardiotoxicity of streptolysin O in rats. Indian J. med. Res. 61, 1584. Game, R. K., Gurra, S., Vnxara, D. R. and Arnr,, P. R. (1976x) Studies on toxic effects of streptolysin O: production of experimental pancarditis in rats . Indian J. Exp. Biol. 14, 242. Game, S., Game, R. K. and VnR~, D. R. (19766) Effect of streptolysin O on myocardial contractility of mammalian heart. LR.C.S. Med. sci. Lib. compend. 4, 120. Gurrx, S., Gurrx, R. K. and SanRrin, K. K. (1976c) Effect of streptolysin O on myocardial contractility of mammalian and amphibian heart. Indian 1. Physiol. Pharmac. 20, 78 . Gyre, S., Gurrx, R. K. and VAxrt~, D. R (19764) Studies on toxic effects of streptolysin O: effect on the contractility of isolated and intact mammalian and amphibian heart. Indian J. Plrysiol. Pharmac. 20, 164. Het,~r, S. P., BIRCHER, R. and D"frrp, E. (1961) The analysis of streptococcal infections-V . Cardiotoxicity of streptolysin O for rabbits in vivo . J. exp. Med. 113, 759. Hwi.eexr, S. P., BuseHSa, R. and D~u.e, E. (1963) Studies on the mechanism of lethal and toxic actions of streptolysin O and the protection by certain antiserotonin drugs. J. Lab. clan. Med. 61, 437. HALPERN, B. N. and RsHAtaN, S. C. (1968) Studies on cardiotoxicity of streptolysin O. Br. J. Pharmac. Chemother. 32, 441 . Kst,r ivex, A., BSRNHIIMEa, A. W., CARLION, A. S. and Fxaent~,x, E. B. (1956) Loss of myocardial contractility in isolated mammalian heart by streptolysin O. J. exp. Med. 104, 361. Rerrz, B. A., Piuaeß, R. D. and F~oex, G. A. (1968) An analysis of the toxic actions of purified stroptolysin O on the isolated heart and separate cardiac tissue of the guinea pig. J. exp. Med. 128, 1401 . Sweas, G. and TRwrs, R. H. (1971) Adrenocortical steroids~ffect on the mesenchymal tissue and antiin8ammatory property . In : 7Tie Pharmacological Basisoj77rerapeutics, 4th edn., p. 1623, (GOODMAN, L. S. and Gn.i .iuw,J , A., Eds.) . London : Macmillan. SpmA, G., Su.emesrenv, Z., HAxRis, T. N. and GixsauRG, I. (1968) Toxic effects induced in rabbits by extracellular products and sonicates of group A streptococci . Proc. Soc. exp. Biol . Med. 127, l196. THOMPSON, A., HALHERT, S. P. and SMrrH, U. (1970) The toxicity of streptolysin O for beating mammalian heart cells in tissue culture. J. exp. Med. 131, 745.