(Scatophagus Argus Linn) Sting Extract on Experimental Animals

Indian Journal of Experimental Biology Vol. 42,May 2004, pp. 461-467

Pharmacological studies on the venomous spotted butterfish (Scatophagus argus Linn) sting extract on experimental animals D Muhuri, SKarmakar, SCDasgupta*, A K Nagchaudhuri** & A Gomes+ Laboratory of Toxi nology and Experimental Pharmacodynamics, Department of Physiology, University of Calcutta, 92, A.P.C. Road, Kolkata 700009, India

Received 9 April 2003; revised 9 February 2004

A sting of the fish S. argus, a venomous edible spotted butterfish, produces tremendous local pain, severe swelling, rise of body temperature, throbbing sensation etc. To establish the pharmacological activities of S. argus sting extract, the pres- ent investigation, was carried out on experimental animals. The LDso of extract was found to be 9.3 mg/kg (iv) in male al- bino mice. The extract showed loss of sensation, urination and salivation in mice. It potentiated pentobarbitone induced sleeping time in male albino mice and produced hypothermia. Extract produced a fall of cat and guinea pig blood pressure, which was completely abolished by mepyramine. It produced a transient reduction of respiratory rate in rat, but decreased respiratory amplitude in cat, which was abolished after vagotomy. On isolated toad heart, the extract increased both the am- plitude and rate of contraction. On isolated guinea pig heart, the sting extract decreased both the rate and amplitude of con- traction leading to cardiac arrest, but it had no effect on isolated guinea pig auricle. The extract produced a reversible block- ade of electrically induced twitch response of isolated chick biventer cervices preparation, but it had no effect on the isolated rat phrenic nerve diaphragm preparation. It produced a slow contractile response on isolated guinea pig ileum, rat uterus and rat fundal strip preparations but produced slow relaxation on isolated rat duodenum preparation. The contractile response on isolated guinea pig ileum and rat fundal strip was antagonised by SCI9220. It did not produce any significant cutaneous haernorrhage in mice and did not produce any haemolysis on saline washed erythrocytes. The sting extract significantly in- creased capillary permeability of guinea pig dorsal flank and produced oedema in mice hind paw.

Key words: Butterfish, Scatophagus argus, Fish envenomation, Fish sting extract IPC Code: Int. C17;A61 K

Fish constitute almost half the number of vertebrates in venorrr', The fish Scatophagus argus locally known on earth 1, and approximately 22000 species of fish are as 'Pyra Chanda' is a venomous edible spotted butter- contained in some 50 orders and 445 families. Of fish (Fig. 1). The geographical distribution of this fish these, more than 200 species of marine fish, including include estuaries of India, Sri Lanka, Philippines, stingrays, scorpionfish, zebrafish, stonefish, weever- China, Taiwan, Queensland (Australia). The sting of fish, toadfish, stargazers and some species of shark, this fish is a common incident among fisherman, that ratfish, catfish, surgeonfish and blenny are known or produces tremendous local pain, rise of body tem- suspected to be venomous".Although only a handful perature, throbbing sensation and severe swelling of of species of venomous fish are thought to be capable of causing human mortality, many other species of fish can produce severe envenomation. The major clinical manifestation of fish envenomation include swelling, increased body temperature, cardiovascular alterations, local necrosis associated with consider- able pain due to pharmacologically active components

*Post Graduate Department of Zoology, Maulana Azad College, Kolkata 700013, India. **Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India. "Correspondent author: Phone: 91-033-2244-4755 Fax: 91-033-2351-9755 & 91-033-2241-3222 Email: [email protected][email protected] Fig. I-The spotted Butterfish Scatophagus argus, Linn. 462 INDIAN J EXP BIOL, MAY 2004

the affected area which keeps away the poor fisher­ Body temperature-Normotherrnic body tempera­ man from their work for few days4. No information is ture was recorded in male albino mice (18-20 g) be­ available on the venom constituent of this fish sting fore and after administration (ip) of extract. Rectal and no specific therapeutic management is known. As temperature was recorded. In control group, extract a result, symptomatic treatment is provided to the was substituted by normal saline. victims. The present investigation is an effort to ex­ Hyperthermic body temperature of rat (160-180 g) 7 plore the pharmacological actions of the Scatophagus was measured after Dandiya and Collumbine • Groups argus Linn, locally known as 'Pyrachanda' sting ex­ of male rats were injected with a 15% suspension of tract in experimental animals in the context of bio­ Brewer's yeast (1 m1I100 g). After 18 hours, the rectal logical action and active constituents. temperature of each animal was measured .. Then ex­ Materials and Methods tract or control saline was administered and tempera­ Anaesthetic ether (Kabra Drugs Ltd, India), acetyl­ ture was recorded up to 3 hr. choline chloride (E. Merck, Germany), atropine sul­ Sleeping time-Pentobarbitone sodium (40 mglkg, ip) phate (Boehringer, Ingelhelm, Germany), brewer's was injected 45 min after administration of either saline yeast (Sigma, USA), chloralose (Sigm"a, USA), cy­ or extract in the control or experimental male albino proheptadine hydrochloride (Merck Sharp Dhome, mice respectively. Time interval between the loss and regaining of righting reflex was measured as sleeping UK), calcium chloride (Merck, India), disodium hy­ 7 drogen phosphate (Merck, India), folin-phenol reagent time • . (SRL, India), glucose (Qualigen, India), histamine Blood pressure--Arterial blood pressure was re­ acid phosphate (Sigma, USA), magnesium chloride corded via indwelling arterial cannula from common (Qualigen, India), mepyramine (May & Baker, UK), carotid artery of male cats (2-3 kg) anaesthetized with methysergide bimaleate (Sandoz, Switzerland), potas­ ether and chloralose (80 mglkg, iv) and from common sium dihydrogen phosphate (Qualigen, India), prosta­ carotid artery of male guinea pigs (200-300 g) anaesthe­ glandin E2 (Sigma, USA), SC 19220 (G.D. Searle & tized with urethan (1.75 g/kg, ip), by a mercury ma­ Co., USA), sodium bicarbonate (Merck, India), so­ nometer on a rotating smoked drum. Spinal cat was pre­ dium pentobarbitone (Loba, India), serotonine creat­ pared by removing second cervical vertebra followed by inine sulphate (E.Merck, Germany), trypan blue transection of exposed spinal cord under standard labo­ (Sigma, USA), and urethane (E. Merck, Germany) ratory conditions. 8 were used. Cat respiration was recorded after Gaddum , before Preparation ofsting extracl---Live fish (S. argus) was and after vagotomy. collected from Canning fish market (West Bengal) dur­ Respiration--Respiration was recorded in urethane ing May to December. The dorsal, pectoral, anal spines (1.75 g/kg, ip) anaesthetized male albino rats (150-200 8 were cut, stored at O°C, immediately transferred to the g) by the method of Gaddum . laboratory and stored at -20°C. The pooled spines were Isolated heart and auricle-lsolated toad heart was ground, homogenized with chilled 0.9% NaCVO.l(M) perfused with frog Ringer's solution through the cannu­ phosphate buffer (PH 7.2) and centrifuged at 10,000 rpm lation of hepatic vein. The contraction was recorded on a at 4°C for 30 min. The supernatant was used as crude rotating drum with the help of a heart lever. venom and stored at 4°C until further use. The sting ex­ Isolated guinea pig heart was prepared after Lan­ tract used as the venom source was expressed in terms of gendorff1 and perfused with oxygenated tyrode solu­ protein equivalent Protein was estimated by the method tion at 37° ± 1°C and contractions were recorded on a 5 of Lowry et al. • rotating smoked drum using a heart lever. Lethality and behavioural changes-LD5o was de­ Isolated guinea pig auricle was prepared as de­ lO termined in male albino mice (20 g) accQrding to the scribed by Burn and suspended (5 ml bath) in oxy­ 6 0 method of Litchfield and Wilcqxon • The sting extract . genated Ringer solution (29 ± 1DC) containing double .. in different doSes (0.2 mVmouse, n=lO.for.each dose) glucose (2 gil). The spontaneous contraction of auri­ was administered through the caudal vein of the cle was recorded on a smoked drum through a Star­ mouse, and mortality was recorded up to 24 hr. ling's lever. Behavioural changes (n=6) were also observed af­ Isolated skeletal muscle-Rat phrenic nerve dia­ l2 ter administration (iv) of the sextract. Animals were phragmll and chick biventer cervices were suspended observed lip to 6 hr. (6 ml bath) in physiological salt solution (NaCI135 rnM, MUHURI et al. " PHARMACOLOGICAL STUDIES ON VENOMOUS BUTIERFISH 463

KCI 5 ruM, CaCh 2 ruM, MgCh 1 mM, NaHC03 15 Body temperature--Extract (2 mg/kg, ip) signifi­ ruM, Na2HP04 1 mM, Glucose 11 mM) gassed with car­ cantly decreased the rectal temperature of normothermic bogen (95% O2 + 5% CO2) at 37° ± 1°C and stimulated male albino mice within 45.50 ± 0.56 min (n=8) and the with a square-wave electronic stimulator (Grass, SD9, normal body temperature returned within 3 hr (control USA, lOY, 0.2 m sec duration, 0.1 Hz). Contractions 37.08° ± 0.16°C, experimental 35.06° ± O.03°C, P < were recorded with Brodie's lever on a smoked Kymo­ 0.(01). graphic paper. In the yeast induced pyretic rats, extract up to a Isolated smooth muscle--Isolated smooth muscle dose of 2 mg/kg, ip did not produce any significant (guinea pig ileum, rat fundal strip, rat uterus, rat duode­ change in body temperature (control 38.51 ± 0.23°C, num) were suspended in oxygenated physiological solu­ experimental 38.42° ± 0.65° C, P > 0.05, n=6). tion and contractions were recorded with a frontal writ­ Sleeping time--Extract (2 mg/kg, ip) significantly I3 14 15 ing lever (Dejalon , Vane , Horton ). potentiated the pentobarbitone induced sleeping time in Haemolysis--Haemolytic activity was tested accord­ male albino mice (control 29.80 ± 0.79 min, experi­ 16 ing to Rothsechild . Citrated blood of different species mental 55.20 ± 0.24 min, n=6, P < 0.(01). was collected and after repeated washings with 0.9% Blood pressure--Extract (0.5 mg/kg, iv, bolus) pro­ saline, a 2% suspension of RBC in saline was prepared. duced a transient fall (19.50 ± 0.60 mmHg, n=7) of cat After 30 min incubation at 37°C with the extract or sa­ blood pressure (Fig.2). The same dose of extract also line, the cells were centrifuged (3000 rpm x 5 min). The produced a fall (15.50 ± 0.42 mrnHg, n=6) of guinea pig extent of haemolysis was estimated in the supernatant blood pressure. In cat and guinea pig, extract (0.5 mg/kg, fluid at optical density of 540 nm. iv, bolus) induced blood pressure fall was 19.66 ± 0.35 Haemorrhagic activity--This was performed on and 15.28 ± 0.20 mmHg (n=6) before mepyrarnine (3 17 shaved albino mice by the modified method of Kondo • mg/kg, iv) treatment. After mepyramine adrllinistration, Extract or saline was injected intradermally into the dor­ same dose of extract did not produce any change in sal skin. After 24 hr, the skin was opened and observed blood pressure in both cat and guinea pig. The hypoten­ for any haemorrhagic lesion. sive response of extract could not abolish before and after spinal transection. Capillary permeability--This was tested on guinea Respiration-Extract (0.5 mg/kg, iv, bolus) produced pig by blue dye extravasation method of Kellet18. Two a transient reduction of respiratory rate (6.67 ± 0.22%, parameters were measured-(a) diameter of the dye ex­ n=6) in rat which returned to normal after 15.25 0.30 travasation areas (corresponding to the site of injection) ± min, but the amplitude remained unaffected. The same on the inner surface of the skin, (b) the amount of dye 9 response was found when the dose of extract was in­ extracted from the skin (Harada et al./ • creased to 1 mg/kg, iv, bolus, where the respiratory rate Paw oedema--For the measurement of paw oedema, was decreased (8.25 ± 0.54%, n=6) which returned to male mice (18-20 g) were used. Paw oedema was in­ normal after 18.28 ± 0.42 min, but no change was ob­ duced by a subplantar injection of 0.02 ml extract, in served in amplitude of respiration up to 3 hours of ob­ 0.002 M phosphate buffer (PH 7.4), into the right hind­ servation. paw. An equal volume of phosphate buffer was injected into the left hind-paw as control. Paw oedema was re­ , 20 corded according to the method of Wang and Teng . 2 ,J All results were expressed as Mean ± SE. The sig­ nificance of the differences between means was de­ termined by Student's 't' test and P values < 0.05 was 100r~ ...... considered as significant. Results 80l v Lethality and behavioural change.Y----LDso of the ex­ H tract was found to be 9.3 mg/kg (iv) in male albino mice. Fig. 2-Effect of S. argus sting extract on cat blood pressure Extract (2 mg/kg, iv) showed loss of sensation, urination [Time scale in min. Vertical scale, blood pressure, mmHg. and salivation. After 2-3 hr, the animals returned to the H= Histamine (2 flglKg, iv), V= Sting extract (0.5 mglKg, normal behaviour. iv), MEP= Mepyramine (3 mg/kg, iv)] 464 INDIAN J EXP BIOL, MAY 2004

In cat, extract (0.5 mg/kg, iv, bolus) produced a did not significantly alter the rate and amplitude of decrease in respiratory amplitude (32.72 ± 0.89%, contraction. n=7), which returned to normal within 1.85 ± 0.26 IsoLated skeletal muscles--Extract up to a dose of 700 min. The extract did .not show any significant effect Ilg/ml it did not produce any significant change on the on the rate of respiration up to 3 hr 'Of observation. electrically induced twitch response of isolated rat The extract-induced respiratory change in· cat was phrenic nerve diaphragm (RPND) preparation. How­ completely abolished after vagotomy. ever, at a dose of 500 Ilg/ml it produced a reversible Isolated heart and auricle-On isolated toad heart, blockade of electrically induced twitch response of iso­ extract (0.8 mg) increased both the amplitude (51.00 ± lated chick biventer cervices (CBC) preparation within 2.25%, n=6) and the rate (32.54 ± 1.58%, n=6) of con­ 15.00 ± 2.32 min, n=6 (Fig. 4). traction. Normal condition returned within 4.83 ± 0.30 Isolated smooth muscles--On isolated guinea pig min (Fig. 3). ileum, extract (80 Ilg/ml) produced a slow contraction On isolated guinea pig heart, extract (0.5 mg) de­ which was not blocked by atropine, methysergide, me­ creased both the rate (84.96±2.50%) and amplitude pyramine pretreament. The contractile effect of extract (84.53 ± 4.27%) of contraction, leading to cardiac was fully antagonised by SC 19220 pretreatment (Fig. arrest within 36.83 ± 1.35 min (n=6). On isolated Sa). On isolated rat duodenum, extract (90 Ilg/ml) pro­ guinea pig auricle, extract up to a dose of (0.5 mg/ml) duced a slow relaxant response. Extract (85 Ilg/ml) pro­ duced a slow contraction on isolated rat uterus prepara­ I 2 tion. The slow contraction was not blocked by cypro­ heptadine. Extract (80 Ilg/ml) produced a slow contrac­ tion on isolated rat fundal strip preparation. The extract­ 84 85 104 106 98 86 82 induced slow contraction was unaffected by methyser­ • • • • • • 0 gide, but was completely antagonised by the prostaglan­ din receptor blocker, SC 19220 (Fig. 5b). The dose­ response relationship of extract was established on iso­ lated guinea pig ileum preparation and was found to be 75Ilg/ml. Haemolysis--Extract up to concentration of 1.2 mg/ml did not produce any significant haemolysis on 2'

Fig. 3-Effect of S. argus sting extract on isolated toad heart. [Time scale in min. Numerical indicates heart rate/min. V= sting extract 800 /lg] ~J • • SC· •• H PG v PG VH

MA~0 · · • sc.. . 5 PO V PGVS kCW Fig. 5--Effect of S. argus sting extract on isolated smooth muscle Fig. 4--Effect of S. argus sting extract on isolated chick biventor preparations [Time scale in min. A= Guinea pig ileum, B= Rat cervices preparation. [Time scale in min. V= sting extract fundal stripe, H= Histamine (2xlO·9 g/ml), PG= Prostaglandin E:! 8 500/lg/ml. K=KCI (1 mg/ml), C= carbachol (Img/ml), W= wash (10.7 g/ml), V~ Sting extract (80 /lg/ml), S= Serotonin (2xlO· 5 (x2 times). g/ml), SC = SC 19220 (3xlO· g/ml »). MUHURI et al. : PHARMACOLOGICAL STUDIES ON VENOMOUS BUITERASH 465 saline washed erythrocytes of mice, rat, guinea pig, rab­ be central. The hypotensive response was completely bit and goat (n=6). antagonized by mepyramine indicating involvement Haemorrhagic activity-Extract up to a dose of 500 of histaminergic receptors or release of histamine like compound by the extract. In vivo responses to piscine Jlg (id, in 0.1 ml) did not produce any significant cuta­ venoms are a function of both their effects on blood neous haemorrhage in mice (n=6). vessels and on the heart. The venom of the catfish Capillary penneability-Extract (100 Jlg in 0.1 ml) H.fossilii2 and venom extracts from the spines of the 23 and normal saline 0.1 ml (control) were injected (id) in freshwater stingray P.motoro produce a marked hy­ guinea pig dorsal flank. The diameter of the area of 'blu­ potensive response in anaesthetized cats and rats re­ 4 ening' as well as the amount of dye were measured. spectively. The venom of the lionfish P. volitani , 25 Both the area of blueing (control 1.5 ± 0.22 mm, extract stonefish S.horrida and the greater weeverfish T.draco26 produce a marked hypotensive response on 17.16 ± 0.40*, n=4, *Prabbits. It was reported that SNTX and 0.62*, n=4, *Pclam lated with C.striatus skin extract, which potentiated isolated hearts and frog bolated atria31 , a rapid pentobarbitone induced sleeping time in male albino chronotropic increase accompanied by an inotropic 21 mice and produced hypothermia . decrease in fish hearts32 and, in rat isolated atria, a The sting extract of S.argus produced a temporary chronotropic increase accompanied by an inotropic 33 hypotensive response in cat and guinea pig, which decrease, then increase . was unaffected after spinal transection. This suggests The sting extract of S.argus produced a reversible that, the seat of hypotensive response was unlikely to blockade of electrically induced twitch response on 466 INDIAN J EXP BIOL, MAY 2004 isolated chick biventer cervices preparation in a dose The skin secretion of the oriental catfish produced 4o dependent manner. The extract did not alter the KCl oedema . The sting extract of S. argus significantly and carbachol induced contraction on chick biventer increased the capillary permeability. It was reported cervices preparation, which indicated that the extract that the venom of the stonefish S. trachynis signifi­ probably acted postsynaptically. The spine tissue ex­ cantly increased capillary permeability in rats41 and tract of lionfish (P. volitans) produced muscle fibrilia­ from the same venom a cytolytic toxin was detected tion in isolated nerve muscle preparation, followed by which exhibited a marked increase in vascular perme­ 31 blockade of neuromuscular transmission • The ability, as shown by the extensive extravasation of the 42 venom of P.canius showed irreversible blockade of Evans blue-plasma albumin complex • This capillary electrically evoked twitches in both the rat phrenic permeability increasing effect may also be involved nerve diaphragm and the chick biventer cervices mus­ with the oedema producing action of the venom/sting 34 35 cle preparation . . It was also reported that the extract. But at present, it is not known whether the venom of the oriental catfish Plotosus lineatus pro­ observed increase in capillary permeability is caused duces severe neurotoxic activity rather potent neuro­ by a direct damaging effect on capillary endothelial 6 muscular activitl • cells or by the release of mediator such as histamine The sting extract produced slow contractile re­ and leukotrines, or by both. Local oedema, redness sponse on isolated smooth muscle preparations, such and pain were the major clinical feature of fish en­ as guinea pig ileum, rat fundal strip and rat uterus. venomation. The clinical finding of the sting of S. But the extract produced relaxation on isolated rat argus (i .e., redness, swelling and pain) are well cor­ duodenum preparation. The contractile response of related with the experimental findings from the pres­ extract on isolated rat uterus was not affected by cy­ ent study. Utilizing these experimental observation, proheptadine but the contraction on guinea pig ileum further experiments are warranted on the development and rat fundal strip was totally antagonized by the of therapeutic antagonists to encounter the enveno­ prostaglandin receptor blocker SC 19220, indicating mation consequences. involvement of prostaglandin like substance(s) pres­ ent in the extract or the extract may have induced the Acknowledgement release of prostaglandin like substances. The slow The authors are grateful to Late Prof. S.c. Lahiri, relaxation produced by the extract on isolated rat for encouragement and valuable suggestions. This duodenum may be due to kinin-like substances pres­ paper is dedicated in his memory. ent in the extract or such substance might have been released by the sting extract of s.argus. Many piscine References venoms have effects on 'ooth vascular and non­ I Nelson J S, Fishes of the world (Wiley Publication, New York) 1984. vascular smooth muscle. It was reported that, solvent 2 Russell FE, Toxic effects of animals toxins, in Casarett and extracts made from the venomous spines of the fresh DouU's toxicology - The basic science of poisons, edited by ' water sting ray P. motoro show a potent cholinergic CD Klaasen (McGraw-Hili Publication, Sydney) 1996. 23 3 Church J E & Hodgson W C, The pharmacological activity effect on the isolated guinea pig ileum . The venoms of fish venoms, Toxicon. 40 (2002) 1083. of the stonefish (S. trachynis) and the soldier fish 4 Muhuri D C, Studies on fish venom (Scatophagus argus). (Gymnapistes marmoratus) also produce contractile Ph.D. Thesis. Jadavpur University. Calcutta (1996). response on isolated guinea pig ileum, which were 5 Lowry 0 H, Rosebrough N J, Farr A L & Randall R J, Pro­ attributed to the venoms containing or releasing both tein measurement with the folin phenol reagent. J Bioi Chern. 37 38 193 (1951) 265. acetylcholine and prostaglandins • • The venoms of 6 Litchfield J T & Wilcoxon F, A simplified method of evalu­ the Indian catfish H. fossilis and P. can ius both pro­ ating dose effects experiments, J Phannacol Exp Ther. 96 duce contractile responses in a number of smooth (1949) 99. muscle preparations, which are attributed to the 7 Dandiya P C & Collumbine H, Studies on Acorus calamus 22 34 (III), some pharmacological action of the volatile oil, J venoms containing or releasing prostaglandins • • A Phannacol Exp Ther. 125 (1959) 353. prostaglandin like substance was also isolated from 8 Gaddum J H, A method for recording the respiration, J the venom of the H. fossilis by solvent extraction and Physiol. 108 (1941) 467. thin layer chromatography39. 9 Langendorff 0, Untersuchngen am ubericbenden sangeticheazen, Pflugers Arch Ges Physiol. 190 (1895) 280. The sting extract of S. argus produced severe 10 Burn J H, . 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