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Sea snake Hydrophis cyanocinctus venom. II. Histopathological changes, induced by a myotoxic phospholipase A2 (PLA2-H1)
Toxicon 38 (2000) 687±705 www.elsevier.com/locate/toxicon
Sea snake Hydrophis cyanocinctus venom. II. Histopathological changes, induced by a myotoxic phospholipase A2 (PLA2-H1) Syed Abid Ali a, b,*, Junaid M. Alam c, Atiya Abbasi a, Zafar H. Zaidi a, Stanka Stoeva b, Wolfgang Voelter b a
International Centre for Chemical Sciences, HEJ Research Institute of Chemistry, University of Karachi, Karachi, 75270, Pakistan b Abteilung fuÈr Physikalische Biochemie, Physiologisch-chemisches Institut der UniversitaÈt, TuÈbingen, Hoppe-Seyler-Straûe 4, D-72076, TuÈbingen, Germany c Department of Biochemistry, Liaquat National Hospital, Karachi, 74800, Pakistan Received 22 October 1998; accepted 15 July 1999
Abstract A toxic phospholipase A2 (PLA2-H1), isolated from the venom of the sea snake Hydrophis cyanocinctus, was tested for its ability to induce myonecrosis and histopathological changes in albino rats and mice. Induction of myonecrosis was demonstrated by their ability to release creatine kinase (CK) from damaged muscle ®bers and direct histopathological examination of the injected muscles (i.m.). PLA2-H1 exhibits intense myonecrosis characterized by the changes including, necrosis and edematous appearance with cellular in®ltrate, vacuolation and degenerated muscle cells with delta lesions and heavy edema in between the cells. No myoglobinuria was noted in any group of animals. The puri®ed PLA2-H1 was also administered intraperitoneally into the experimental animals and tissue samples were taken at several time intervals. Light
Abbreviations: PLA2, phospholipase A2; i.p., intraperitoneal; H & E, hematoxylin and eosin; LD50, half lethal dose; id., intradermal; im., intramuscular; SDS±PAGE, sodium dodecyl sulfate±polyacrylamide gel electrophoresis; MALDI MS, matrix-assisted laser desorption ionization mass spectrometery; RP HPLC, reverse phase high performance liquid chromatography; CK, creatine kinase. * Correspondening author. Present address: Abteilung fuÈr Physikalische Biochemie des Physiologischchemischen, Instituts der UniversitaÈt TuÈbingen, Hoppe-Seyler-Straûe 4, D-72076 TuÈbingen, Germany. Tel.: +49-07071-2975336; fax: 07071-293348. E-mail address: [email protected] (S.A. Ali). 0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 9 ) 0 0 1 8 4 - 1
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microscopic examination of the kidney sections revealed severe damage, evident by focal tubular necrosis, complete disquamation of epithelial lining and epithelial degeneration of tubules in all test animals. Light micrographs of liver sections after 24 h of injection shows fatty in®ltration in parenchyma and squashed hepatocytes, while after 48 h, fatty vacuolation of parenchyma in a generalized pattern was observed. Furthermore, sections of the lungs of the same group of animals (48 h) show dilated bronchia and marked in®ltration of in¯ammatory cells within alveoli. Our results suggest that the puri®ed PLA2H1 induced moderate myotoxicity in muscles and mild histopathological changes in other vital organs without myoglobinuria. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Sea snake; Hydrophis cyanocinctus; Hydrophiinae; Venom; Myotoxins; Phospholipase A2; Histopathological changes
1. Introduction Acute renal dysfunction (Soe-Soe et al., 1990; Zimmerman et al., 1992; Gopalakrishnakone et al., 1993), myonecrosis (Arroyo et al., 1987), skeletal muscle damage (Preston et al., 1990; Chen et al., 1993), pneumonitis and hepatitis (Alam et al., 1995) are the few common features resulting from accidental and experimental snake envenoming (Marsden and Reid, 1961; Reid, 1961). These pathological conditions have been ascribed to the eects of necrotic (Onrat et al., 1993) and cytotoxic components (Selistre et al., 1990; Ponraj and Gopalakrishnakone, 1995; Gopalakrishnakone, 1997) in snake venom (short/long chain neurotoxins, cyto/cardiotoxins, necrotic and hemorrhagic factors/toxins and several enzymes), including important and the major class of enzyme, the phospholipases (Nakai et al., 1995; Huang and Gopalakrishnakone, 1996; for review see Stocker, 1990). It has been reported that venom phospholipases, specially phospholipases A2 (EC 3.1.1.4), act on phospholipids of the hemostatic system, on thrombocytes to inhibit adhesion and aggregation (Ouyang et al., 1987; Kini and Evans, 1990), red cells to cause hemolysis (Condrea, 1979), cardiac cells to induce cardiotoxicity (Huang et al., 1993), muscle cells to cause myonecrosis (Mebs and Samejima, 1986; Mebs and Ownby, 1990; DõÂ az et al., 1992; GutieÂrrez and Lomonte, 1995), nerve cells to prevent neurotransmission and cause cell damage in general (Kini and Iwanga, 1986; Harris, 1991; Chen et al., 1994; Tsai et al., 1995). Despite their wide variety of pathophysiological/pharmacological eects, either dependent or independent of their catalytic activity (Rosenberg, 1986; Kini and Evans, 1989), PLA2(s) from snake venoms show close structural similarities and constitute a large family of a homologous (14-kDa) class of proteins (for review see Arni and Ward, 1996). Sea snakes are classi®ed in the family Hydrophiinae and Laticaudinae and are widely distributed in the tropical Paci®c and Indian ocean. In the coastal water of the Pakistan region, including Karachi and Makran coasts, thirteen species of sea snakes have been reported (Iat, 1988) of which the six major species, including
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Hydrophis cyanocinctus, belong to a single genus, Hydrophis. Sea snake poisoning is a rare, but potentially serious hazard of the marine environment, as the venoms contain potent toxins and also phospholipases (Tan and Ponnudurai, 1991) being more lethal than venoms of many terrestrial snakes (Tu, 1987; Ali et al., 1998). Envenomations by sea snakes, although being generally considered as non-fatal and most of the time asymtomatic, cause paralysis, dyspea, muscle spasms, myonecrosis, hepato- and nephrotoxicity (Minton, 1974; Yang and Lee, 1978; Acott and Williamson, 1996). We have noted intense myonecrosis induced by a single dose of a myotoxic phospholipase A2 (PLA2-H1), isolated from the sea snake Hydrophis cyanocinctus venom. Associated acute nephritis, hepatitis and mild pneumonitis were also apparent in all test groups of animals. The present communication describes the histopathological changes induced by PLA2-H1 in dierent organs, pathogenesis, and possible mechanisms of such myonecrotic, nephrotic and hepatic syndromes have been discussed.
2. Materials and methods 2.1. Venom collection and puri®cation of phospholipase A2 Sea snake species of Hydrophis cyanocinctus were collected from eustarian water of Karachi coast (Pakistan). Snakes were identi®ed by the Zoological Survey Department, Karachi. Venom from live snakes was squeezed out manually, lyophilized immediately and stored at ÿ208C till further use (Ali et al., 1998). The isolation/puri®cation and characterization of the PLA2-H1, as well as its chemical and biological properties was the subject of separate communications and described elsewhere (Ali et al., 1998, 1999). Brie¯y, puri®cation of a toxic PLA2 component (H1) from sea snake Hydrophis cyanocinctus venom was achieved by single step RP-HPLC on a Nucleosil 7C18 column. A total of 25 fractions were separated, three of which had phospholipase A2 activity. Puri®ed PLA2-H1 is a 13588 Da single polypeptide chain (as determined by SDS±PAGE and MALDI MS), enzymatically active and a toxic component of the whole venom with a LD50 (i.p.) of 0.045 mg/kg (body weight in albino rats). Furthermore, PLA2-H1 also produces edema and indirect hemolytic activities (Ali et al., 1999). 2.2. Animals Fifty albino rats, weighing 100±150 g, of either sex and eighty Male Swiss Wistar mice (approximately weight 30 g) were used. The animals were housed ®ve per cage in a room with 12:12 h light±dark cycle at a room temperature (258C) and allowed to consume water and laboratory prepared feed ad libitum.
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2.3. Assay for generalized histopathological changes The rats were divided into ®ve groups (I±V). Group I (10 rats) given physiological saline (i.p.) serve as control. Group II and III (10 rats each) were injected (i.p.) with 3.0 mg of puri®ed PLA2-H1 in 100 ml saline and sacri®ced after 24 and 48 h, respectively. Group IV and V (10 rats each) received 6.0 mg of PLA2H1 in 100 ml saline (i.p.) and were sacri®ced at 24 and 48 h, respectively. Mice were divided in three groups (VI±VIII, n = 6 each). Mice in groups VI and VII received 1.5 mg (i.p.) of PLA2-H1 in 50 ml saline and killed after 24 and 48 h, respectively. Group VIII received 50 ml physiological saline and served as control. LD50 in mice was also determined in a separate group of animals (test; n = 15, control; n = 5) by i.p. injection of 5±10 mg of PLA2-H1 in 0.9% NaCl (w/v). LD50 was determined at ®ve dose levels and observed during 24 h period. 2.4. Preparation for histopathological examination The animals were autopsied and all internal organs were macroscopically examined and ®xed in 7% formalin. Liver, kidneys, lungs and spleen were embedded in paran and sliced. The slides for light microscopy were stained with hematoxylin and eosin (H & E). A section of right thigh muscle from groups VI± VIII of mice was also excised and stained with H & E and Masson's trichrome stains. All preparations were made and analyzed as described earlier (Alam and Qasim, 1997; Alam et al., 1993). Kidney sections from rat and mice were also stained with Massons's trichrome stain to monitor myoglobin casts (Ponraj and Gopalakrishnakone, 1995). Blood was also collected from group I through VIII and the creatine kinase (CK) level was determined to examine tissue destruction (see below). 2.5. Determination of the ability of PLA2-H1 to induce myonecrosis and myoglobinuria 2.5.1. Enzyme assay The liberation of creatine kinase from damaged muscle cells was followed by using the enzymatic UV method (Deutsche Gesellschaft fuÈr Klinische Chemie) as described in the assay manual (Boehringer-Mannheim, Germany) to measure enzyme activity in plasma. Male Swiss Wistar mice (approximately 30 g) were injected in the right thigh (gastrocnemius muscle) with 1.5 mg of PLA2-H1 in 25 ml 0.9% sodium chloride (w/v). The PLA2-H1 injected mice were sacri®ced at various time intervals at t = 30 min, 1 h, 6 h, 12 h, 18 h, 24 h, 48 h, 72 h and 6 days (groups A to I, respectively (n = 4) and blood was collected from the abdominal cava and the plasma separated by centrifugation at 15,000 rpm for 3 min at room temperature. Control animals (n = 6) received 0.9% sodium chloride only (Mancuso et al., 1995; Ponraj and Gopalakrishnakone, 1995).
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2.5.2. Assay for myonecrosis For the myonecrosis, induced by PLA2-H1, a small portion of the central region of gastrocnemius muscle was excised from all groups (groups A to I and control) and post-®xed overnight in 10% formalin, dehydrated through a series of ethanol treatments and embedded in wax. Sections of 5 mm thickness were cut (Shandon AS 325, USA), stained with H & E and examined under the light microscope (Nikon, Japan). Areas exhibiting pathology were photographed with a Konica (black and white) ®lm. Kidneys were also removed from all groups, processed as described above and stained with Masson's trichrome stain. Whereas, lungs, hearts, livers and spleens were removed, processed similarly and stained with H & E. 2.5.3. Assay for myoglobinuria The myoglobinuria assay was performed by placing the animals on white ®lter paper after injection at de®ned time intervals (see Section 2.5.1). In positive cases, the paper should stain red or dark brown when the animals urinate (Ponraj and Gopalakrishnakone, 1995). 3. Results 3.1. Lethality Puri®ed PLA2-H1 was found to be highly toxic to albino rats (LD50 i.p. 0.045 mg/kg) and mice (LD50 i.p. 0.036 mg/kg). Puri®cation resulted in an 18-fold increase in toxicity over the crude venom (LD50 i.p. 0.805 mg/kg in rats and 0.730 mg/kg in mice). 3.2. Generalized pathological ®ndings After i.p. injection, PLA2-H1 was highly toxic to animals causing reduced mobility within a few hours. Generalized paralysis appeared after 10±12 h in rats and 5±6 h in mice. Most of the animals did not survive after 35 h in all test groups, except in groups II and III, where 4 and 3 out of 10 rats survived up to 40 h, respectively. Macroscopic examination of autopsied organs in, both, rats and mice showed congested lungs, livers and swollen kidneys. Changes in the wet weight of aected kidneys were also noticed which was slightly higher as compared to the control group. In addition, kidneys were tender on touching. Likewise, slightly swollen and discoloured liver was noted, specially from those animals surviving upto 40 h. No visible change was observed in lungs, spleens and hearts. Notably, hearts and spleens showed no pathological changes in any group. Intramuscular injection in mice also did not induce any notable change in all organs except the area of muscle injected. Morphologically, mild changes were only seen in kidneys of groups sacri®ced after 48 h. Light microscopic examination of kidney sections, from all test groups showed
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generalized tubular degeneration (Fig. 1B, C). However, tubular degeneration was more prominent in groups surviving up to 40 h, specially exhibiting a profound degenerated epithelial lining of tubules. Complete disquamation of epithelial lining was also noted with sporadic focal tubular necrosis (Fig. 1B, C). Marked nephrotoxicity in the present study was possibly due to the fact that the kidneys are the main ®ltration organs of the blood and thus the main source of elimination of toxaemia which is experimentally induced by PLA2 administration. Kidney sections, stained with Masson's trichrome stain, do not reveal the presence of any myoglobin casts, but do exhibit similar pathological ®ndings in both animal groups, i.e. mice and rats. Microscopic examination of liver and lungs showed considerable changes in animals surviving upto 40 h. Marked fatty changes in liver were noted in all subjects (Fig. 2B, C). Hepatic injury is also characterized by degenerated hepatocytes and identi®ed by swollen edematous hepatocytes with clumped cytoplasm and large clear spaces (Fig. 2C). Furthermore, lung sections showed in®ltration of in¯ammatory cells within bronchus alveoli and dilation of bronchus (Fig. 3B). None of the muscle sections after i.p. injection, either stained with H & E or Masson's trichrome stains showed any signi®cant pathological changes.
Fig. 1. (A) Light micrograph of kidney from control rats receiving 100 ml of normal saline (i.p.). The section shows normal kidney tubules with intact epithelial linings (arrow head), magni®cation 350 H & E stain. (B) Photomicrograph from test group V receiving 6.0 mg of PLA2-H1 (i.p.). The section shows focal tubular necrosis (arrow head), complete disquamation of epithelial lining (arrows) and epithelial degeneration of tubules (double arrow heads), magni®cation 350 H & E stain. (C) Photomicrograph from test group V, receiving 6.0 mg of PLA2-H1 (i.p.) and showing marked epithelial degeneration of tubules (arrow heads), magni®cation 1400 H & E stain.
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Fig. 1 (continued)
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However, analysis of plasma for creatine kinase liberation by lysed tissues and cells revealed a several-fold increase in its level, both, at 24 and 48 h in all groups II through to VII (data not shown). 3.3. Myonecrosis The maximum peak of CK liberation from damaged muscle, induced by PLA2H1 in mice by intramuscular injection (i.m.) in groups A to I, was obtained at dierent time intervals. PLA2-H1-induced gradual increase in CK liberation within 24 h. Three peaks of high CK liberation were detected, the ®rst peak at t = 12 h, the second at t = 18 h and the third at t = 24 h. However, after 48 h, the CK level gradually normalized (Fig. 4). The muscle tissue from the control group was normal (Fig. 5A), whereas the tissue from test groups D to F (12 h, 18 h and 24 h, respectively) exhibited intense myonecrosis throughout the muscle sections. The changes include necrosis and edematous appearance with cellular in®ltrate (Fig. 5B), vacuolation and degenerated muscle cells with delta lesions and heavy edema in between the cells (Fig. 5C, D). None of the tissue of the animals in groups A to C (30 min, 1 h, 6 h, respectively) showed prominent myonecrosis. Similarly, there
Fig. 2. (A) Light micrograph of liver from control group I receiving 100 ml saline (i.p.). The section shows normal hepatocytes (arrowheads) and uniform parenchyma, magni®cation 750 H & E stain. (B) Photomicrograph from test group IV receiving 6.0 mg of puri®ed PLA2-H1 and sacri®ced after 24 h. The section shows generalized fatty in®ltration in parenchyma (arrowheads) and squashed hepatocytes (arrow), magni®cation 350 H & E stain. (C) Photomicrograph from test group V, receiving 6.0 mg of puri®ed PLA2-H1 and sacri®ced after 48 h. The section shows fatty vacuolation of parenchyma (arrowsheads) in a generalized pattern, magni®cation 750 H & E stain.
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Fig. 2 (continued)
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Fig. 3. (A) Light micrograph of lung from control normal bronchus (arrowhead) and intact alveoli receiving 6.0 mg PLA2-H1 (i.p.) and sacri®ced after and marked in®ltration of in¯ammatory cells within
group receiving 100 ml saline (i.p.). Section shows (arrow). (B) Photomicrograph of test group V 48 h. Section shows dilated bronchus (arrowhead) alveoli (arrows) magni®cation 350 H & E stain.
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Fig. 4. Myotoxic activity of phospholipase A2 (PLA2-H1) in mice after intramuscular (i.m.) administration followed in terms of plasma creatine kinase liberation.
are no signi®cant myonecrotic areas observed in groups G to I (48 h, 72 h, 6 days, respectively). There were no pathological changes observed in the lungs, spleen and heart in any of the groups examined. However, the kidney sections of the groups sacri®ced at 48 and 72 h (groups G and H, respectively) showed mild tubular degeneration. Furthermore, fatty in®ltration in liver was observed, but only in animals of the group sacri®ced after 24 h, probably due to the lipolytic action of PLA2-H1. No myoglobinuria was noted in any of the groups of animals during the whole period of experimentation.
4. Discussion We have examined the generalized histopathological and myonecrotic activities of a puri®ed PLA2-H1 from the sea snake Hydrophis cyanocinctus venom in rats and mice. The lethal dose was determined to be 0.045 mg/kg in rats and 0.036 mg/ kg in mice which makes PLA2-H1 a potent lethal toxin as compared to its nontoxic component PLA2-H2 (Ali et al., 1999). It has been reported that the LD50 value can distinguish two major groups of phospholipases, those with LD50(s) less than 1 mg/kg and those with LD50(s) greater than 1 mg/kg (Mebs and Ownby, 1990). The phospholipases of the former group represent the most lethal snake venom components. The results of our studies indicate that PLA2-H1 causes pathological changes to kidneys, livers and lungs after i.p. injection, whereas additionally myonecrosis was induced with mild nephritis and hepatitis after i.m. injection. Interestingly, no myonecrosis was noted in groups injected intraperitoneally with PLA2-H1. Likewise, no myoglobinuria was detected in any
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Fig. 5. Light micrograph of muscles (H & E stain) from mice 24 h after (i.m.) injection of 1.5 mg of myotoxic PLA2-H1 from sea snake Hydrophis cyanocinctus venom. (A) Control tissue taken from mice injected with 25 ml of 0.9% NaCl, showing the normal appearance of muscle cells. (B) Muscle cells 24 h after (i.m.) injection of 1.5 mg PLA2-H1 showing the celluar in®ltrate (double arrowheads), edema (arrowhead) and necrotic cells (arrows). (C) Aected muscle cells containing vacuoles (arrows), degenerated muscle cells (double arrowheads) and marked edema (arrowhead). (D) Aected muscle cells with vacuoles (arrowhead) with few delta lesions (arrows) and edema (double arrowheads).
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Fig. 5 (continued)
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of the animal groups. Masson's trichrome staining of kidneys also did not reveal any myoglobin cast in kidney sections examined. However, induced nephrotoxicity was possibly due to the fact that the kidneys are the main ®ltration organ of blood and thus the main source of elimination of toxaemia. During the elimination of PLA2-H1 by kidney compartments (viz, tubules and glomerulus) which depend on blood supply, PLA2-H1 caused tubular and interstitial disorders through its cytotoxic potencies. We have noticed marked tubular degeneration and necrosis in our study, as described in the preceding section, having a generalized feature and possibly caused by a generalized origin. However, various types of nephritis were characterized by several basic tissue reactions, of which hypercellularity or in¯ammatory disease are more common (Cotran et al., 1994). The in¯ammatory disease is characterized by cellular proliferation of mesangial and endothelial cells and leukocytic in®ltration. We have observed mild hypercellularity of lymphocytic origin in all kidney sections, both, at 24 and 48 h intervals in groups receiving i.p. treatment (Fig. 1B, C). These in¯ammatory cells release speci®c mediators (viz. histamine, 5-HT, oxygen metabolites, and nitric oxide) upon activation, which cause vasodilation, endothelial damage and cytotoxicity. Other mechanisms may also be responsible in contributing towards nephrotic injury, e.g. injury of epithelial cells, renal ablation, glomerulopathy or interstitial in¯ammation. Of these four, epithelial cell injury is often the result from antibody±antigen reactions (Emancipator, 1992), toxins (Ashkenazi, 1993) or/and cytokines (Border and Nobel, 1993). Previously, necrotic debris and degenerated tubular epithelium was also noted in kidneys of mice injected with myotoxic PLA2 (Ponraj and Gopalakrishnakone, 1995). Crude snake venoms also induce tubular degeneration as reported for rabbits by the injection of Vipera russelli's venom (Soe-Soe et al., 1990). Acute renal tubular degeneration and proliferative glomerulonephritis was also noticed in mice by the venom of the sea snake Apiysurus laevis (Zimmerman et al., 1992). Terao et al. (1994) also reported morphological changes in kidney tubules induced by an algal toxin, cylindrospermopsin, from Umezakia natans, including single cell necrosis in distal and proximal urinary tubules. Furthermore, earlier studies by Reid and co-workers (Marsden and Reid, 1961; Reid, 1961) on sea snake envenomation victims revealed renal damage, specially tubular necrosis, edematous interstitial necrosis and, both, diuse and focal cellular in®ltration. Regarding the basis of hepatic injury as seen in our study (Fig. 2B, C), several reactions have been documented preceding the hepatic injury regardless of the nature of the causative agents (Cotran et al., 1994). These reactions are necrosis, degeneration, in¯ammation, regeneration and ®brosis. In necrosis, which is mediated by toxic agents, isolated hepatocytes round up to form shrunken, pyknotic and intensely eosinolytic councilman bodies. We have noted sporadic hydropic degeneration, where hepatocytes osmotically swell and rupture (Fig. 2C). Centrilobular necrosis, observed in few slides (though not very frequent), have also been reported as a characteristic injury and occur due to many drugs and toxins (Kaplowitz, 1992). Previously, we have also noticed similar eects in guinea pigs induced by Hydrophis cyanocinctus whole venom (Alam and Qasim, 1993;
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Alam et al., 1995), eventually caused by the presence of some other potent nonenzymatic toxins, e.g. neurotoxins and cyto/cardiotoxins (unpublished data). Furthermore, earlier reports have also shown centrilobular degeneration and cellular in®ltration of portal areas in the liver, suggested to be due to the direct eect of the whole venom on the hepatic system (Marsden and Reid, 1961). In the present studies lung sections showed in®ltration of in¯ammatory cells within bronchus alveoli and dilation of bronchus (Fig. 3B). However, no conclusive mechanism was reported yet for pneumonitis occurring due to sea snake envenomation, even though edema and in¯ammatory changes in bronchi was noted in the early 60 s by Marsden and Reid (1961). Therefore, pneumonitis observed in the present studies was not due to the direct action of the venom or its components, but probably due to factors related to renal dysfunction which induces bronchopneumonia and acute and chronic in¯ammatory response to broncospasm. The myotoxicity induced by PLA2-H1 in mice was examined by two methods, i.e. determination of creatine kinase, released from damaged muscle ®ber, and the direct histological examination of the injected muscle (Mancuso et al., 1995). PLA2-H1 induced the highest CK release by i.m. injection and less by i.p. injection (Fig. 4). These results suggest a correlation between the levels of CK and the intensity of myonecrosis. Maximum CK liberation peaks were observed at t = 12 h, 18 h and 24 h. Marked release of CK was also noted at t = 6 h and 48 h. However, the CK level is 10 times lower than observed in former treatments. Previously, CK liberation, as induced by venoms from the snakes of the Genus Micrururus, was determined at t = 3 h and t = 6 h (Barros et al., 1994). In the present study it was found that the maximum peak of CK liberation did not occur always at such a short span of time as observed with the venoms from the snakes of the Genus Micrururus. It is indicated that the occurrence of the CK liberation peaks most likely correspond to the direct myotoxic eects of PLA2 in the body (Barros et al., 1994). The type of myonecrosis that we have observed, closely relates to those described earlier by several myotoxic PLA2s (Fohlman and Eaker, 1977; Arroyo et al., 1987; Johnson and Ownby, 1993; Ponraj and Gopalakrishnakone, 1995; Huang and Gopalakrishnakone, 1996; Ownby et al., 1997) and also myotoxins devoid of detectable phospholipase activity (GutieÂrrez et al., 1989; GutieÂrrez et al., 1992; Lomonte and GutieÂrrez, 1989; Johnson and Ownby, 1993; Mancuso et al., 1995; Soares et al., 1998). However, PLA2-H1 is a relatively slow acting myotoxin than those reported earlier, indicating variations in the myotoxic potencies of dierent phospholipases. Previously, Huang and Gopalakrishnakone (1996) reported on dierences in the potencies of myotoxins, although having homologous structures. For example the PLA2(s) from crotalid venom produces myonecrotic changes in muscles, but the minimum dose to induce morphological changes may vary. Similarly, Trimeresurus ¯avovidis and Bothrops asper myotoxins are about 5±10 times more active than those from Agkistrodon venoms (Fohlman and Eaker, 1977; Mebs and Samejima, 1986). The absence of any myotoxic activity of PLA2-H1 in lungs, hearts and spleens
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might be caused by the non-susceptibility of the muscle and tissue towards the enzyme. This is well supported from another PLA2 from the sea snake Enhydrina schistosa which is a highly active myotoxin, but shows no eect on cardiac and intrafusal muscles (Brooks et al., 1987; Huang and Gopalakroshnakone, 1996). In contrast, PLA2 from Pseudechis colletti produces pathological changes in myocardium and skeletal muscle (Weinstein et al., 1992). Thus, from these investigations the conclusions must be drawn that dierent PLA2(s) cause speci®c actions on dierent muscles and tissues.
Acknowledgements We express our gratitute to the Deutscher Akademischer Austauschdienst (DAAD-Bonn, Germany) for a research scholarship (41-HP-cr-gb) to S.A.Ali. Acknowledgements are due for Dr. Naveen Faridi (Department of Histopathology, Liaquat National Hospital, Karachi) for her encouragement and advice of the present study. Financial aid by Fonds der Chemischen Industrie is also greatly acknowledged.
References Acott, C., Williamson, J., 1996. Sea snake. In: Williamson, J.A., Fenner, P.J., Burnett, J.W., Rifkin, J.F. (Eds.), Venomous and Poisonous Marine Animals: A Medical and Biological Handbook. UNSW Press, Sydney, pp. 396±402. Alam, J.M., Qasim, R., 1993. Changes in serum components induced by venoms of marine animals. Pak. J. Pharmac. Sci. 6, 81±87. Alam, J.M., Qasim, R., 1997. Isolation of cytotoxic compounds from marine animals. I-Hepatotoxin PvHTxIIE from shell®sh Perna viridis (green mussels). Pak. J. Zool. 29, 61±75. Alam, J.M., Alam, S.M., Qasim, R., 1993. Pathogenesis of the morphological lesions in various organs in guniea pigs produced by cytotoxic compounds from marine animals. Pak. J. Pathol. 4, 95±99. Alam, J.M., Qasim, R., Alam, S.M., 1995. Eects of drugs against cytotoxic compounds from marine animals IV-hepatotoxic activity of sea snake Hydrophis cyanocinctus venom. Pak. J. Pharmaceut. Sci. 8, 63±68. Ali, S.A., Alam, J.M., Zaidi, Z.H., Abbasi, A., 1998. Isolation and characterization of a toxic phospholipase A2 from sea snake (Hydrophis cyanocinctus ) venom. Pak. J. Zool. 30, 289±294. Ali, S.A., Alam, J.M., Stoeva, S., SchuÈtz, J., Abbasi, A., Zaidi, Z.H., Voelter, W., 1999. Sea snake Hydrophis cyanocinctus venom. I. Puri®cation, characterization and N-terminal sequence of two phospholipases A2. Toxicon 37, 1505±1520. Arni, R.K., Ward, R.J., 1996. Phospholipase A2-A structural review. Toxicon 34, 827±841. Arroyo, O., Rosso, J.P., Vargas, O., GutieÂrrez, J.M., Cerdas, L., 1987. Skeletal muscle necrosis induced by a phospholipase A2 isolated from the venom of the coral snake Micrurus nigrocinctus nigrocinctus. Comp. Biochem. Physiol. 87, 949±952. Ashkenazi, S., 1993. Role of bacterial cytotoxins in hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Annu. Rev. Med. 44, 11±18. Barros, A.C.S., Fernandes, D.P., Ferreira de, L.C. L., Dos, Santos M.C., 1994. Local eects induced by venoms from ®ve species of genus Micrurus sp. (Coral snakes). Toxicon 32, 445±452. Border, W.A., Nobel, N.A., 1993. Cytokines in kidney disease: the role of transforming growth factorbeta (b ). Am. J. Kidney Dis. 22, 105±113.
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Brooks, G., Torres, L., Gopalakrishnakone, P., Duchen, L., 1987. Eects of phospholipase of Enhydrina schistosa venom on nerve, motor end-plate and muscle of the mouse. Q. J. Exp. Physiol. 72, 571±591. Chen, S.Z.J., Gopalakrishnakone, P., Gwee, M.C.E., 1993. Light and electron microscopic studies of the eects of the venom from Pseudechis australis in the murine soleus muscel. In: Tan, N.H., Oo, S.L., Thambyrajah, V., Azila, N. (Eds.), Advances in Venom and Toxin Research: Animal, Plant and Microbial Toxins. Malaysian Society on Toxinology, Kuala Lumpur, pp. 234±239. Chen, L-N., Liu, C-S., Chang, C-C., 1994. Isolation and characterization of a toxic phospholipase A2 from the venom of the Taiwan habu (Trimeresurus mucrosquamatus ). Biotechnol. Appl. Biochem. 19, 61±73. Condrea, E., 1979. Hemolytic eects of snake venoms. In: Lee, C.Y. (Ed.), Snake venoms, Handbook of Experimental Pharmacology, vol. 52. Springer-Verlag, Berlin, pp. 448±462. Cotran, R.S., Kumar, V., Robbins, S.L., 1994. The Kidneys. In: Cotran, R.S., Kumar, V., Robbins, S.L. (Eds.), Robbins pathological basis of disease, 5th ed. Saunders, Philadelphia, pp. 927±989. DõÂ az, C., GutieÂrrez, J.M., Lomonte, B., 1992. Isolation and characterization of basic myotoxic phospholipase A2 from Bothrops godmani (Godman's pit viper) snake venom. Arch. Biochem. Biophys. 298, 135±142. Emancipator, S.N., 1992. Primary and secondary forms of IgA nephritis. In: Heptinstall, R.H. (Ed.), Pathology of Kidney. Little Brown, Boston, pp. 389±476. Fohlman, J., Eaker, D., 1977. Isolation and characterization of a lethal myotoxic phospholipase from the venom of common sea snake Enhydrina schistosa causing myoglobinuria in mice. Toxicon 15, 385±393. Gopalakrishnakone, P., Chen, S.Z.J., Gwee, M.C.E., 1993. Ultrastructural changes induced by the myoglobinuria-producing venom from Pseudechis australis in mice kidney. In: Tan, N.H., Oo, S.L., Thambyrajah, V., Azila, N. (Eds.), Advances in Venom and Toxin Research: Animal, Plant, and Microbial Toxins. Malaysian Society on Toxinology, Kuala Lumpur, pp. 129±134. Gopalakrishnakone, P., 1997. Myotoxic phospholipases from snake venoms: general myoglobinuria and local myonecrotic toxins. Toxicon 35, 476. GutieÂrrez, J.M., Lomonte, B., 1995. Phospholipases A2 myotoxins from Bothrops snake venoms: Review article. Toxicon 33, 1405±1424. GutieÂrrez, J.M., Chaves, F., Gene, J.A., Lomonte, B., Camacho, Z., Schosinsky, K., 1989. Myonecrosis induced by a basic myotoxin isolated from the venom of the snake Bothrops asper. Exp. Molec. Path. 52, 25±36. GutieÂrrez, J.M., Rojas, G., Jorge de Silva Jr, N., Nunez, J., 1992. Experimental myonecrosis induced by the venoms of South American Micrurus (Coral snake). Toxicon 30, 1299±1302. Harris, J.B., 1991. Phospholipases in snake venoms and their eects on nerve and muscle. In: Harvey, A.L. (Ed.), Snake Toxins. Pergamon Press, New York, pp. 91±129. Huang, M-Z., Wang, Q.C., Liu, G.F., 1993. Eects of an acidic phospholipase A2 puri®ed from Ophiophagus hannah (king cobra) venom on rat heart. Toxicon 31, 627±635. Huang, M-Z., Gopalakrishnakone, P., 1996. Pathalogical changes induced by an acidic phospholipase A2 from Ophiophagus hannah venom on the heart and skeletal muscle of mice after systemic injection. Toxicon 34, 201±211. Iat, F., 1988. Marine snakes of Pakistan. Rec. Zool. Surv. Pak. 11, 1±56. Johnson, E.K., Ownby, C.L., 1993. Isolation of a myotoxin from the venom of Agkistrodon contortrix laticinctus (broad-banded copper head) and pathogenesis of myonecrosis induced it in mice. Toxicon 31, 245±255. Kaplowitz, N., 1992. Drug metabolism and hepatotoxicity. In: Kaplowitz, N. (Ed.), Liver and Billary Diseases. Williams & Wilkins, Baltimore, pp. 82±97. Kini, R.M., Iwanga, S., 1986. Structure-function relationships of phospholipases I: Prediction of presynaptic neurotoxicity. Toxicon 24, 527±541. Kini, R.M., Evans, H.J., 1989. A model to explain the pharmacological eects of snake venom phospholipases A2. Toxicon 27, 613±635. Kini, R.M., Evans, H.J., 1990. Eects of snake venom proteins on blood platelets. Toxicon 28, 1387± 1422.
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S.A. Ali et al. / Toxicon 38 (2000) 687±705
Lomonte, B., GutieÂrrez, J.M., 1989. A new muscle damaging toxin, myotoxin II, from the venom of the snake Bothrops asper (terciopelo). Toxicon 27, 725±733. Mancuso, L.C., Correa, M.M., Vieira, C.A., Cunha, O.A.B., Lachat, J-J., Selistre de Araujo, H.S., Ownby, C.L., Giglio, J.R., 1995. Fractionation of Bothrops pirajai snake venom: isolation and characterization of piratoxin-I, a new myotoxic protein. Toxicon 33, 615±626. Marsden, A.T.H., Reid, H.A., 1961. Pathology of sea snake poisoning. Brit. Med. J. 1, 1290±1293. Mebs, D., Samejima, Y., 1986. Isolation and characterization of myotoxic phospholipase A2 from crotalid venoms. Toxicon 24, 161±168. Mebs, D., Ownby, C.L., 1990. Myotoxic components of snake venoms: their biochemical and biological activities. Pharmac. Ther. 48, 223±236. Minton Jr, S.A., 1974. Snake and snake venoms. In: Kugelmass, N. (Ed.), Venom Diseases. Thomas, Illinois, pp. 107±124. Nakai, M., Nakashima, K.I., Ogawa, T., Shimohigashi, Y., Hattori, S., Chang, C.C., Ohno, M., 1995. Puri®cation and primary structure of a myotoxic lysine-49 phospholipase A2 with low lipolytic activity from Trimeresurus gramineus venom. Toxicon 33, 1469±1478. Onrat, D., Pakmanee, N., Thammapalert, W., Tampitag, S., 1993. Characterization of local tissue necrotic factor(s) fraction I and XIII from Siamese Cobra venom. In: Tan, N.H., Oo, S.L., Thambyrajah, V., Azila, N. (Eds.), Advances in Venom and Toxin Research: Animal, Plant and Microbial Toxins. Malaysian Society on Toxinology, Kuala Lumpur, pp. 141±146. Ouyang, C., Teng, C.M., Huang, T.F., 1987. Characterization of snake venom principles aecting blood coagulation and platelet aggregation. Asia Paci®c J. Pharm. 2, 169±179. Ownby, C.L., Collerg, T.R., White, S.P., 1997. Isolation, characterization and crystallization of a phospholipases A2 myotoxin from the venom of the prairie rattle snake (Crotalus viridis viridis ). Toxicon 35, 111±124. Ponraj, D., Gopalakrishnakone, P., 1995. Morphological changes induced by a generalized myotoxin (myoglobinuria-inducing toxin) from the venom of Pseudechis australis (king brown snake) in skeletal muscle and kidney of mice. Toxicon 33, 1453±1467. Preston, S.A., Davis, C.E., Harris, J.B., 1990. The assessment of muscel ®bre loss after the injection of the venom of Notechis scutatus (Australian Tiger Snake). Toxicon 28, 201±214. Reid, H.A., 1961. Myoglobinuria and sea snake bite poisoning. Brit. Med. J. 1, 1284±1289. Rosenberg, P.R., 1986. The relationship between enzymatic activity and pharmacological properties of phospholipases in natural poisons. In: Harris, J.B. (Ed.), Natural Toxins. Clarendon Press, Oxford, pp. 129±174. Selistre, H.S., Queiroz, L.S., Cunha, O.A.B., De Souza, G.E.P., Giglio, J.R., 1990. Isolation and characterization of hemorrhagic, myonecrotic and edema-inducing toxins from Bothrops insularis (jararaca ilhoa) venom. Toxicon 28, 261±273. Soares, A.M., Rodrigues, V.M., Homsi-Brandeburgo, M.I., Toyama, M.H., Lombardi, F.R., Arni, R.K., Giglio, J.R., 1998. A rapid procedure for the isolation of lys-49 myotoxin II from Bothrops moojeni (Caissaca) venom: biochemical characterization, crystallization, myotoxic and edematogenic activity. Toxicon 36, 503±514. Soe-Soe, Than-Than, Khin-Ei-Han, 1990. The nephrotoxic action of Russell's viper (Vipera russelli ) venom. Toxicon 28, 461±467. Stocker, K.F., 1990. Composition of snake venoms. In: Stocker, K.F. (Ed.), Medical Use of Snake Venom Proteins. CRC Press, Boca Raton, FL, pp. 33±56. Tan, N-H., Ponnudurai, G.A., 1991. Comparative study of the biological properties of some sea snake venoms. Comp. Biochem. Physiol. 99, 351±354. Terao, K., Ohmori, S., Igarashi, K., Ohtani, I., Watanabe, M.F., Harada, K.I., Ito, E., Watanabe, M., 1994. Electron microscopic studies on experimental poisoning in mice induced by cylindrospermopsin isolated from blue-green alga Umezakia natans. Toxicon 32, 833±843. Tsai, I-H., Lu, P-J., Wang, Y-M., Ho, C-L., Liaw, L-L., 1995. Molecular cloning and characterization of a neurotoxic phospholipase A2 from the venom of Taiwan habu (Trimeresurus mucrosquamatus ). Biochem. J. 311, 895±900. Tu, A.T., 1987. Biotoxicology of sea snake venoms. Annal. Emerg. Med. 16, 149±154. Weinstein, S.A., Bell, R.C., Brown II, C.R., Fletcher, J.E., Smith, L.A., 1992. Pathologic changes
S.A. Ali et al. / Toxicon 38 (2000) 687±705
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induced by phospholipase A2 isolated from the venom of Collett's snake, Pseudechis colletti: a light and electron microscopic study. Toxicon 30, 171±185. Yang, T.Y., Lee, C.Y., 1978. Pharmacological studies on the venom of a sea snake Hydrophis cyanocinctus. In: Rosenberg, P. (Ed.), Toxins, Animals, Plant and Microbial. Pergamon Press, New York, pp. 261±292. Zimmerman, S.E., Heatwole, H.H., Andreopoulos, P.C., Yong, L.C.J., 1992. Proliferative glomerulonephritis in mice induced by sea snake (Aipysurus laevis ) venom. Exp. Toxicol. Pathol. 44, 294±300.
Sea snake Hydrophis cyanocinctus venom. II. Histopathological changes, induced by a myotoxic phospholipase A2 (PLA2-H1) Syed Abid Ali a, b,*, Junaid M. Alam c, Atiya Abbasi a, Zafar H. Zaidi a, Stanka Stoeva b, Wolfgang Voelter b a
International Centre for Chemical Sciences, HEJ Research Institute of Chemistry, University of Karachi, Karachi, 75270, Pakistan b Abteilung fuÈr Physikalische Biochemie, Physiologisch-chemisches Institut der UniversitaÈt, TuÈbingen, Hoppe-Seyler-Straûe 4, D-72076, TuÈbingen, Germany c Department of Biochemistry, Liaquat National Hospital, Karachi, 74800, Pakistan Received 22 October 1998; accepted 15 July 1999
Abstract A toxic phospholipase A2 (PLA2-H1), isolated from the venom of the sea snake Hydrophis cyanocinctus, was tested for its ability to induce myonecrosis and histopathological changes in albino rats and mice. Induction of myonecrosis was demonstrated by their ability to release creatine kinase (CK) from damaged muscle ®bers and direct histopathological examination of the injected muscles (i.m.). PLA2-H1 exhibits intense myonecrosis characterized by the changes including, necrosis and edematous appearance with cellular in®ltrate, vacuolation and degenerated muscle cells with delta lesions and heavy edema in between the cells. No myoglobinuria was noted in any group of animals. The puri®ed PLA2-H1 was also administered intraperitoneally into the experimental animals and tissue samples were taken at several time intervals. Light
Abbreviations: PLA2, phospholipase A2; i.p., intraperitoneal; H & E, hematoxylin and eosin; LD50, half lethal dose; id., intradermal; im., intramuscular; SDS±PAGE, sodium dodecyl sulfate±polyacrylamide gel electrophoresis; MALDI MS, matrix-assisted laser desorption ionization mass spectrometery; RP HPLC, reverse phase high performance liquid chromatography; CK, creatine kinase. * Correspondening author. Present address: Abteilung fuÈr Physikalische Biochemie des Physiologischchemischen, Instituts der UniversitaÈt TuÈbingen, Hoppe-Seyler-Straûe 4, D-72076 TuÈbingen, Germany. Tel.: +49-07071-2975336; fax: 07071-293348. E-mail address: [email protected] (S.A. Ali). 0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 9 ) 0 0 1 8 4 - 1
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microscopic examination of the kidney sections revealed severe damage, evident by focal tubular necrosis, complete disquamation of epithelial lining and epithelial degeneration of tubules in all test animals. Light micrographs of liver sections after 24 h of injection shows fatty in®ltration in parenchyma and squashed hepatocytes, while after 48 h, fatty vacuolation of parenchyma in a generalized pattern was observed. Furthermore, sections of the lungs of the same group of animals (48 h) show dilated bronchia and marked in®ltration of in¯ammatory cells within alveoli. Our results suggest that the puri®ed PLA2H1 induced moderate myotoxicity in muscles and mild histopathological changes in other vital organs without myoglobinuria. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Sea snake; Hydrophis cyanocinctus; Hydrophiinae; Venom; Myotoxins; Phospholipase A2; Histopathological changes
1. Introduction Acute renal dysfunction (Soe-Soe et al., 1990; Zimmerman et al., 1992; Gopalakrishnakone et al., 1993), myonecrosis (Arroyo et al., 1987), skeletal muscle damage (Preston et al., 1990; Chen et al., 1993), pneumonitis and hepatitis (Alam et al., 1995) are the few common features resulting from accidental and experimental snake envenoming (Marsden and Reid, 1961; Reid, 1961). These pathological conditions have been ascribed to the eects of necrotic (Onrat et al., 1993) and cytotoxic components (Selistre et al., 1990; Ponraj and Gopalakrishnakone, 1995; Gopalakrishnakone, 1997) in snake venom (short/long chain neurotoxins, cyto/cardiotoxins, necrotic and hemorrhagic factors/toxins and several enzymes), including important and the major class of enzyme, the phospholipases (Nakai et al., 1995; Huang and Gopalakrishnakone, 1996; for review see Stocker, 1990). It has been reported that venom phospholipases, specially phospholipases A2 (EC 3.1.1.4), act on phospholipids of the hemostatic system, on thrombocytes to inhibit adhesion and aggregation (Ouyang et al., 1987; Kini and Evans, 1990), red cells to cause hemolysis (Condrea, 1979), cardiac cells to induce cardiotoxicity (Huang et al., 1993), muscle cells to cause myonecrosis (Mebs and Samejima, 1986; Mebs and Ownby, 1990; DõÂ az et al., 1992; GutieÂrrez and Lomonte, 1995), nerve cells to prevent neurotransmission and cause cell damage in general (Kini and Iwanga, 1986; Harris, 1991; Chen et al., 1994; Tsai et al., 1995). Despite their wide variety of pathophysiological/pharmacological eects, either dependent or independent of their catalytic activity (Rosenberg, 1986; Kini and Evans, 1989), PLA2(s) from snake venoms show close structural similarities and constitute a large family of a homologous (14-kDa) class of proteins (for review see Arni and Ward, 1996). Sea snakes are classi®ed in the family Hydrophiinae and Laticaudinae and are widely distributed in the tropical Paci®c and Indian ocean. In the coastal water of the Pakistan region, including Karachi and Makran coasts, thirteen species of sea snakes have been reported (Iat, 1988) of which the six major species, including
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Hydrophis cyanocinctus, belong to a single genus, Hydrophis. Sea snake poisoning is a rare, but potentially serious hazard of the marine environment, as the venoms contain potent toxins and also phospholipases (Tan and Ponnudurai, 1991) being more lethal than venoms of many terrestrial snakes (Tu, 1987; Ali et al., 1998). Envenomations by sea snakes, although being generally considered as non-fatal and most of the time asymtomatic, cause paralysis, dyspea, muscle spasms, myonecrosis, hepato- and nephrotoxicity (Minton, 1974; Yang and Lee, 1978; Acott and Williamson, 1996). We have noted intense myonecrosis induced by a single dose of a myotoxic phospholipase A2 (PLA2-H1), isolated from the sea snake Hydrophis cyanocinctus venom. Associated acute nephritis, hepatitis and mild pneumonitis were also apparent in all test groups of animals. The present communication describes the histopathological changes induced by PLA2-H1 in dierent organs, pathogenesis, and possible mechanisms of such myonecrotic, nephrotic and hepatic syndromes have been discussed.
2. Materials and methods 2.1. Venom collection and puri®cation of phospholipase A2 Sea snake species of Hydrophis cyanocinctus were collected from eustarian water of Karachi coast (Pakistan). Snakes were identi®ed by the Zoological Survey Department, Karachi. Venom from live snakes was squeezed out manually, lyophilized immediately and stored at ÿ208C till further use (Ali et al., 1998). The isolation/puri®cation and characterization of the PLA2-H1, as well as its chemical and biological properties was the subject of separate communications and described elsewhere (Ali et al., 1998, 1999). Brie¯y, puri®cation of a toxic PLA2 component (H1) from sea snake Hydrophis cyanocinctus venom was achieved by single step RP-HPLC on a Nucleosil 7C18 column. A total of 25 fractions were separated, three of which had phospholipase A2 activity. Puri®ed PLA2-H1 is a 13588 Da single polypeptide chain (as determined by SDS±PAGE and MALDI MS), enzymatically active and a toxic component of the whole venom with a LD50 (i.p.) of 0.045 mg/kg (body weight in albino rats). Furthermore, PLA2-H1 also produces edema and indirect hemolytic activities (Ali et al., 1999). 2.2. Animals Fifty albino rats, weighing 100±150 g, of either sex and eighty Male Swiss Wistar mice (approximately weight 30 g) were used. The animals were housed ®ve per cage in a room with 12:12 h light±dark cycle at a room temperature (258C) and allowed to consume water and laboratory prepared feed ad libitum.
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2.3. Assay for generalized histopathological changes The rats were divided into ®ve groups (I±V). Group I (10 rats) given physiological saline (i.p.) serve as control. Group II and III (10 rats each) were injected (i.p.) with 3.0 mg of puri®ed PLA2-H1 in 100 ml saline and sacri®ced after 24 and 48 h, respectively. Group IV and V (10 rats each) received 6.0 mg of PLA2H1 in 100 ml saline (i.p.) and were sacri®ced at 24 and 48 h, respectively. Mice were divided in three groups (VI±VIII, n = 6 each). Mice in groups VI and VII received 1.5 mg (i.p.) of PLA2-H1 in 50 ml saline and killed after 24 and 48 h, respectively. Group VIII received 50 ml physiological saline and served as control. LD50 in mice was also determined in a separate group of animals (test; n = 15, control; n = 5) by i.p. injection of 5±10 mg of PLA2-H1 in 0.9% NaCl (w/v). LD50 was determined at ®ve dose levels and observed during 24 h period. 2.4. Preparation for histopathological examination The animals were autopsied and all internal organs were macroscopically examined and ®xed in 7% formalin. Liver, kidneys, lungs and spleen were embedded in paran and sliced. The slides for light microscopy were stained with hematoxylin and eosin (H & E). A section of right thigh muscle from groups VI± VIII of mice was also excised and stained with H & E and Masson's trichrome stains. All preparations were made and analyzed as described earlier (Alam and Qasim, 1997; Alam et al., 1993). Kidney sections from rat and mice were also stained with Massons's trichrome stain to monitor myoglobin casts (Ponraj and Gopalakrishnakone, 1995). Blood was also collected from group I through VIII and the creatine kinase (CK) level was determined to examine tissue destruction (see below). 2.5. Determination of the ability of PLA2-H1 to induce myonecrosis and myoglobinuria 2.5.1. Enzyme assay The liberation of creatine kinase from damaged muscle cells was followed by using the enzymatic UV method (Deutsche Gesellschaft fuÈr Klinische Chemie) as described in the assay manual (Boehringer-Mannheim, Germany) to measure enzyme activity in plasma. Male Swiss Wistar mice (approximately 30 g) were injected in the right thigh (gastrocnemius muscle) with 1.5 mg of PLA2-H1 in 25 ml 0.9% sodium chloride (w/v). The PLA2-H1 injected mice were sacri®ced at various time intervals at t = 30 min, 1 h, 6 h, 12 h, 18 h, 24 h, 48 h, 72 h and 6 days (groups A to I, respectively (n = 4) and blood was collected from the abdominal cava and the plasma separated by centrifugation at 15,000 rpm for 3 min at room temperature. Control animals (n = 6) received 0.9% sodium chloride only (Mancuso et al., 1995; Ponraj and Gopalakrishnakone, 1995).
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2.5.2. Assay for myonecrosis For the myonecrosis, induced by PLA2-H1, a small portion of the central region of gastrocnemius muscle was excised from all groups (groups A to I and control) and post-®xed overnight in 10% formalin, dehydrated through a series of ethanol treatments and embedded in wax. Sections of 5 mm thickness were cut (Shandon AS 325, USA), stained with H & E and examined under the light microscope (Nikon, Japan). Areas exhibiting pathology were photographed with a Konica (black and white) ®lm. Kidneys were also removed from all groups, processed as described above and stained with Masson's trichrome stain. Whereas, lungs, hearts, livers and spleens were removed, processed similarly and stained with H & E. 2.5.3. Assay for myoglobinuria The myoglobinuria assay was performed by placing the animals on white ®lter paper after injection at de®ned time intervals (see Section 2.5.1). In positive cases, the paper should stain red or dark brown when the animals urinate (Ponraj and Gopalakrishnakone, 1995). 3. Results 3.1. Lethality Puri®ed PLA2-H1 was found to be highly toxic to albino rats (LD50 i.p. 0.045 mg/kg) and mice (LD50 i.p. 0.036 mg/kg). Puri®cation resulted in an 18-fold increase in toxicity over the crude venom (LD50 i.p. 0.805 mg/kg in rats and 0.730 mg/kg in mice). 3.2. Generalized pathological ®ndings After i.p. injection, PLA2-H1 was highly toxic to animals causing reduced mobility within a few hours. Generalized paralysis appeared after 10±12 h in rats and 5±6 h in mice. Most of the animals did not survive after 35 h in all test groups, except in groups II and III, where 4 and 3 out of 10 rats survived up to 40 h, respectively. Macroscopic examination of autopsied organs in, both, rats and mice showed congested lungs, livers and swollen kidneys. Changes in the wet weight of aected kidneys were also noticed which was slightly higher as compared to the control group. In addition, kidneys were tender on touching. Likewise, slightly swollen and discoloured liver was noted, specially from those animals surviving upto 40 h. No visible change was observed in lungs, spleens and hearts. Notably, hearts and spleens showed no pathological changes in any group. Intramuscular injection in mice also did not induce any notable change in all organs except the area of muscle injected. Morphologically, mild changes were only seen in kidneys of groups sacri®ced after 48 h. Light microscopic examination of kidney sections, from all test groups showed
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generalized tubular degeneration (Fig. 1B, C). However, tubular degeneration was more prominent in groups surviving up to 40 h, specially exhibiting a profound degenerated epithelial lining of tubules. Complete disquamation of epithelial lining was also noted with sporadic focal tubular necrosis (Fig. 1B, C). Marked nephrotoxicity in the present study was possibly due to the fact that the kidneys are the main ®ltration organs of the blood and thus the main source of elimination of toxaemia which is experimentally induced by PLA2 administration. Kidney sections, stained with Masson's trichrome stain, do not reveal the presence of any myoglobin casts, but do exhibit similar pathological ®ndings in both animal groups, i.e. mice and rats. Microscopic examination of liver and lungs showed considerable changes in animals surviving upto 40 h. Marked fatty changes in liver were noted in all subjects (Fig. 2B, C). Hepatic injury is also characterized by degenerated hepatocytes and identi®ed by swollen edematous hepatocytes with clumped cytoplasm and large clear spaces (Fig. 2C). Furthermore, lung sections showed in®ltration of in¯ammatory cells within bronchus alveoli and dilation of bronchus (Fig. 3B). None of the muscle sections after i.p. injection, either stained with H & E or Masson's trichrome stains showed any signi®cant pathological changes.
Fig. 1. (A) Light micrograph of kidney from control rats receiving 100 ml of normal saline (i.p.). The section shows normal kidney tubules with intact epithelial linings (arrow head), magni®cation 350 H & E stain. (B) Photomicrograph from test group V receiving 6.0 mg of PLA2-H1 (i.p.). The section shows focal tubular necrosis (arrow head), complete disquamation of epithelial lining (arrows) and epithelial degeneration of tubules (double arrow heads), magni®cation 350 H & E stain. (C) Photomicrograph from test group V, receiving 6.0 mg of PLA2-H1 (i.p.) and showing marked epithelial degeneration of tubules (arrow heads), magni®cation 1400 H & E stain.
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Fig. 1 (continued)
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However, analysis of plasma for creatine kinase liberation by lysed tissues and cells revealed a several-fold increase in its level, both, at 24 and 48 h in all groups II through to VII (data not shown). 3.3. Myonecrosis The maximum peak of CK liberation from damaged muscle, induced by PLA2H1 in mice by intramuscular injection (i.m.) in groups A to I, was obtained at dierent time intervals. PLA2-H1-induced gradual increase in CK liberation within 24 h. Three peaks of high CK liberation were detected, the ®rst peak at t = 12 h, the second at t = 18 h and the third at t = 24 h. However, after 48 h, the CK level gradually normalized (Fig. 4). The muscle tissue from the control group was normal (Fig. 5A), whereas the tissue from test groups D to F (12 h, 18 h and 24 h, respectively) exhibited intense myonecrosis throughout the muscle sections. The changes include necrosis and edematous appearance with cellular in®ltrate (Fig. 5B), vacuolation and degenerated muscle cells with delta lesions and heavy edema in between the cells (Fig. 5C, D). None of the tissue of the animals in groups A to C (30 min, 1 h, 6 h, respectively) showed prominent myonecrosis. Similarly, there
Fig. 2. (A) Light micrograph of liver from control group I receiving 100 ml saline (i.p.). The section shows normal hepatocytes (arrowheads) and uniform parenchyma, magni®cation 750 H & E stain. (B) Photomicrograph from test group IV receiving 6.0 mg of puri®ed PLA2-H1 and sacri®ced after 24 h. The section shows generalized fatty in®ltration in parenchyma (arrowheads) and squashed hepatocytes (arrow), magni®cation 350 H & E stain. (C) Photomicrograph from test group V, receiving 6.0 mg of puri®ed PLA2-H1 and sacri®ced after 48 h. The section shows fatty vacuolation of parenchyma (arrowsheads) in a generalized pattern, magni®cation 750 H & E stain.
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Fig. 2 (continued)
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Fig. 3. (A) Light micrograph of lung from control normal bronchus (arrowhead) and intact alveoli receiving 6.0 mg PLA2-H1 (i.p.) and sacri®ced after and marked in®ltration of in¯ammatory cells within
group receiving 100 ml saline (i.p.). Section shows (arrow). (B) Photomicrograph of test group V 48 h. Section shows dilated bronchus (arrowhead) alveoli (arrows) magni®cation 350 H & E stain.
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Fig. 4. Myotoxic activity of phospholipase A2 (PLA2-H1) in mice after intramuscular (i.m.) administration followed in terms of plasma creatine kinase liberation.
are no signi®cant myonecrotic areas observed in groups G to I (48 h, 72 h, 6 days, respectively). There were no pathological changes observed in the lungs, spleen and heart in any of the groups examined. However, the kidney sections of the groups sacri®ced at 48 and 72 h (groups G and H, respectively) showed mild tubular degeneration. Furthermore, fatty in®ltration in liver was observed, but only in animals of the group sacri®ced after 24 h, probably due to the lipolytic action of PLA2-H1. No myoglobinuria was noted in any of the groups of animals during the whole period of experimentation.
4. Discussion We have examined the generalized histopathological and myonecrotic activities of a puri®ed PLA2-H1 from the sea snake Hydrophis cyanocinctus venom in rats and mice. The lethal dose was determined to be 0.045 mg/kg in rats and 0.036 mg/ kg in mice which makes PLA2-H1 a potent lethal toxin as compared to its nontoxic component PLA2-H2 (Ali et al., 1999). It has been reported that the LD50 value can distinguish two major groups of phospholipases, those with LD50(s) less than 1 mg/kg and those with LD50(s) greater than 1 mg/kg (Mebs and Ownby, 1990). The phospholipases of the former group represent the most lethal snake venom components. The results of our studies indicate that PLA2-H1 causes pathological changes to kidneys, livers and lungs after i.p. injection, whereas additionally myonecrosis was induced with mild nephritis and hepatitis after i.m. injection. Interestingly, no myonecrosis was noted in groups injected intraperitoneally with PLA2-H1. Likewise, no myoglobinuria was detected in any
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Fig. 5. Light micrograph of muscles (H & E stain) from mice 24 h after (i.m.) injection of 1.5 mg of myotoxic PLA2-H1 from sea snake Hydrophis cyanocinctus venom. (A) Control tissue taken from mice injected with 25 ml of 0.9% NaCl, showing the normal appearance of muscle cells. (B) Muscle cells 24 h after (i.m.) injection of 1.5 mg PLA2-H1 showing the celluar in®ltrate (double arrowheads), edema (arrowhead) and necrotic cells (arrows). (C) Aected muscle cells containing vacuoles (arrows), degenerated muscle cells (double arrowheads) and marked edema (arrowhead). (D) Aected muscle cells with vacuoles (arrowhead) with few delta lesions (arrows) and edema (double arrowheads).
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Fig. 5 (continued)
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of the animal groups. Masson's trichrome staining of kidneys also did not reveal any myoglobin cast in kidney sections examined. However, induced nephrotoxicity was possibly due to the fact that the kidneys are the main ®ltration organ of blood and thus the main source of elimination of toxaemia. During the elimination of PLA2-H1 by kidney compartments (viz, tubules and glomerulus) which depend on blood supply, PLA2-H1 caused tubular and interstitial disorders through its cytotoxic potencies. We have noticed marked tubular degeneration and necrosis in our study, as described in the preceding section, having a generalized feature and possibly caused by a generalized origin. However, various types of nephritis were characterized by several basic tissue reactions, of which hypercellularity or in¯ammatory disease are more common (Cotran et al., 1994). The in¯ammatory disease is characterized by cellular proliferation of mesangial and endothelial cells and leukocytic in®ltration. We have observed mild hypercellularity of lymphocytic origin in all kidney sections, both, at 24 and 48 h intervals in groups receiving i.p. treatment (Fig. 1B, C). These in¯ammatory cells release speci®c mediators (viz. histamine, 5-HT, oxygen metabolites, and nitric oxide) upon activation, which cause vasodilation, endothelial damage and cytotoxicity. Other mechanisms may also be responsible in contributing towards nephrotic injury, e.g. injury of epithelial cells, renal ablation, glomerulopathy or interstitial in¯ammation. Of these four, epithelial cell injury is often the result from antibody±antigen reactions (Emancipator, 1992), toxins (Ashkenazi, 1993) or/and cytokines (Border and Nobel, 1993). Previously, necrotic debris and degenerated tubular epithelium was also noted in kidneys of mice injected with myotoxic PLA2 (Ponraj and Gopalakrishnakone, 1995). Crude snake venoms also induce tubular degeneration as reported for rabbits by the injection of Vipera russelli's venom (Soe-Soe et al., 1990). Acute renal tubular degeneration and proliferative glomerulonephritis was also noticed in mice by the venom of the sea snake Apiysurus laevis (Zimmerman et al., 1992). Terao et al. (1994) also reported morphological changes in kidney tubules induced by an algal toxin, cylindrospermopsin, from Umezakia natans, including single cell necrosis in distal and proximal urinary tubules. Furthermore, earlier studies by Reid and co-workers (Marsden and Reid, 1961; Reid, 1961) on sea snake envenomation victims revealed renal damage, specially tubular necrosis, edematous interstitial necrosis and, both, diuse and focal cellular in®ltration. Regarding the basis of hepatic injury as seen in our study (Fig. 2B, C), several reactions have been documented preceding the hepatic injury regardless of the nature of the causative agents (Cotran et al., 1994). These reactions are necrosis, degeneration, in¯ammation, regeneration and ®brosis. In necrosis, which is mediated by toxic agents, isolated hepatocytes round up to form shrunken, pyknotic and intensely eosinolytic councilman bodies. We have noted sporadic hydropic degeneration, where hepatocytes osmotically swell and rupture (Fig. 2C). Centrilobular necrosis, observed in few slides (though not very frequent), have also been reported as a characteristic injury and occur due to many drugs and toxins (Kaplowitz, 1992). Previously, we have also noticed similar eects in guinea pigs induced by Hydrophis cyanocinctus whole venom (Alam and Qasim, 1993;
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Alam et al., 1995), eventually caused by the presence of some other potent nonenzymatic toxins, e.g. neurotoxins and cyto/cardiotoxins (unpublished data). Furthermore, earlier reports have also shown centrilobular degeneration and cellular in®ltration of portal areas in the liver, suggested to be due to the direct eect of the whole venom on the hepatic system (Marsden and Reid, 1961). In the present studies lung sections showed in®ltration of in¯ammatory cells within bronchus alveoli and dilation of bronchus (Fig. 3B). However, no conclusive mechanism was reported yet for pneumonitis occurring due to sea snake envenomation, even though edema and in¯ammatory changes in bronchi was noted in the early 60 s by Marsden and Reid (1961). Therefore, pneumonitis observed in the present studies was not due to the direct action of the venom or its components, but probably due to factors related to renal dysfunction which induces bronchopneumonia and acute and chronic in¯ammatory response to broncospasm. The myotoxicity induced by PLA2-H1 in mice was examined by two methods, i.e. determination of creatine kinase, released from damaged muscle ®ber, and the direct histological examination of the injected muscle (Mancuso et al., 1995). PLA2-H1 induced the highest CK release by i.m. injection and less by i.p. injection (Fig. 4). These results suggest a correlation between the levels of CK and the intensity of myonecrosis. Maximum CK liberation peaks were observed at t = 12 h, 18 h and 24 h. Marked release of CK was also noted at t = 6 h and 48 h. However, the CK level is 10 times lower than observed in former treatments. Previously, CK liberation, as induced by venoms from the snakes of the Genus Micrururus, was determined at t = 3 h and t = 6 h (Barros et al., 1994). In the present study it was found that the maximum peak of CK liberation did not occur always at such a short span of time as observed with the venoms from the snakes of the Genus Micrururus. It is indicated that the occurrence of the CK liberation peaks most likely correspond to the direct myotoxic eects of PLA2 in the body (Barros et al., 1994). The type of myonecrosis that we have observed, closely relates to those described earlier by several myotoxic PLA2s (Fohlman and Eaker, 1977; Arroyo et al., 1987; Johnson and Ownby, 1993; Ponraj and Gopalakrishnakone, 1995; Huang and Gopalakrishnakone, 1996; Ownby et al., 1997) and also myotoxins devoid of detectable phospholipase activity (GutieÂrrez et al., 1989; GutieÂrrez et al., 1992; Lomonte and GutieÂrrez, 1989; Johnson and Ownby, 1993; Mancuso et al., 1995; Soares et al., 1998). However, PLA2-H1 is a relatively slow acting myotoxin than those reported earlier, indicating variations in the myotoxic potencies of dierent phospholipases. Previously, Huang and Gopalakrishnakone (1996) reported on dierences in the potencies of myotoxins, although having homologous structures. For example the PLA2(s) from crotalid venom produces myonecrotic changes in muscles, but the minimum dose to induce morphological changes may vary. Similarly, Trimeresurus ¯avovidis and Bothrops asper myotoxins are about 5±10 times more active than those from Agkistrodon venoms (Fohlman and Eaker, 1977; Mebs and Samejima, 1986). The absence of any myotoxic activity of PLA2-H1 in lungs, hearts and spleens
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might be caused by the non-susceptibility of the muscle and tissue towards the enzyme. This is well supported from another PLA2 from the sea snake Enhydrina schistosa which is a highly active myotoxin, but shows no eect on cardiac and intrafusal muscles (Brooks et al., 1987; Huang and Gopalakroshnakone, 1996). In contrast, PLA2 from Pseudechis colletti produces pathological changes in myocardium and skeletal muscle (Weinstein et al., 1992). Thus, from these investigations the conclusions must be drawn that dierent PLA2(s) cause speci®c actions on dierent muscles and tissues.
Acknowledgements We express our gratitute to the Deutscher Akademischer Austauschdienst (DAAD-Bonn, Germany) for a research scholarship (41-HP-cr-gb) to S.A.Ali. Acknowledgements are due for Dr. Naveen Faridi (Department of Histopathology, Liaquat National Hospital, Karachi) for her encouragement and advice of the present study. Financial aid by Fonds der Chemischen Industrie is also greatly acknowledged.
References Acott, C., Williamson, J., 1996. Sea snake. In: Williamson, J.A., Fenner, P.J., Burnett, J.W., Rifkin, J.F. (Eds.), Venomous and Poisonous Marine Animals: A Medical and Biological Handbook. UNSW Press, Sydney, pp. 396±402. Alam, J.M., Qasim, R., 1993. Changes in serum components induced by venoms of marine animals. Pak. J. Pharmac. Sci. 6, 81±87. Alam, J.M., Qasim, R., 1997. Isolation of cytotoxic compounds from marine animals. I-Hepatotoxin PvHTxIIE from shell®sh Perna viridis (green mussels). Pak. J. Zool. 29, 61±75. Alam, J.M., Alam, S.M., Qasim, R., 1993. Pathogenesis of the morphological lesions in various organs in guniea pigs produced by cytotoxic compounds from marine animals. Pak. J. Pathol. 4, 95±99. Alam, J.M., Qasim, R., Alam, S.M., 1995. Eects of drugs against cytotoxic compounds from marine animals IV-hepatotoxic activity of sea snake Hydrophis cyanocinctus venom. Pak. J. Pharmaceut. Sci. 8, 63±68. Ali, S.A., Alam, J.M., Zaidi, Z.H., Abbasi, A., 1998. Isolation and characterization of a toxic phospholipase A2 from sea snake (Hydrophis cyanocinctus ) venom. Pak. J. Zool. 30, 289±294. Ali, S.A., Alam, J.M., Stoeva, S., SchuÈtz, J., Abbasi, A., Zaidi, Z.H., Voelter, W., 1999. Sea snake Hydrophis cyanocinctus venom. I. Puri®cation, characterization and N-terminal sequence of two phospholipases A2. Toxicon 37, 1505±1520. Arni, R.K., Ward, R.J., 1996. Phospholipase A2-A structural review. Toxicon 34, 827±841. Arroyo, O., Rosso, J.P., Vargas, O., GutieÂrrez, J.M., Cerdas, L., 1987. Skeletal muscle necrosis induced by a phospholipase A2 isolated from the venom of the coral snake Micrurus nigrocinctus nigrocinctus. Comp. Biochem. Physiol. 87, 949±952. Ashkenazi, S., 1993. Role of bacterial cytotoxins in hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Annu. Rev. Med. 44, 11±18. Barros, A.C.S., Fernandes, D.P., Ferreira de, L.C. L., Dos, Santos M.C., 1994. Local eects induced by venoms from ®ve species of genus Micrurus sp. (Coral snakes). Toxicon 32, 445±452. Border, W.A., Nobel, N.A., 1993. Cytokines in kidney disease: the role of transforming growth factorbeta (b ). Am. J. Kidney Dis. 22, 105±113.
S.A. Ali et al. / Toxicon 38 (2000) 687±705
703
Brooks, G., Torres, L., Gopalakrishnakone, P., Duchen, L., 1987. Eects of phospholipase of Enhydrina schistosa venom on nerve, motor end-plate and muscle of the mouse. Q. J. Exp. Physiol. 72, 571±591. Chen, S.Z.J., Gopalakrishnakone, P., Gwee, M.C.E., 1993. Light and electron microscopic studies of the eects of the venom from Pseudechis australis in the murine soleus muscel. In: Tan, N.H., Oo, S.L., Thambyrajah, V., Azila, N. (Eds.), Advances in Venom and Toxin Research: Animal, Plant and Microbial Toxins. Malaysian Society on Toxinology, Kuala Lumpur, pp. 234±239. Chen, L-N., Liu, C-S., Chang, C-C., 1994. Isolation and characterization of a toxic phospholipase A2 from the venom of the Taiwan habu (Trimeresurus mucrosquamatus ). Biotechnol. Appl. Biochem. 19, 61±73. Condrea, E., 1979. Hemolytic eects of snake venoms. In: Lee, C.Y. (Ed.), Snake venoms, Handbook of Experimental Pharmacology, vol. 52. Springer-Verlag, Berlin, pp. 448±462. Cotran, R.S., Kumar, V., Robbins, S.L., 1994. The Kidneys. In: Cotran, R.S., Kumar, V., Robbins, S.L. (Eds.), Robbins pathological basis of disease, 5th ed. Saunders, Philadelphia, pp. 927±989. DõÂ az, C., GutieÂrrez, J.M., Lomonte, B., 1992. Isolation and characterization of basic myotoxic phospholipase A2 from Bothrops godmani (Godman's pit viper) snake venom. Arch. Biochem. Biophys. 298, 135±142. Emancipator, S.N., 1992. Primary and secondary forms of IgA nephritis. In: Heptinstall, R.H. (Ed.), Pathology of Kidney. Little Brown, Boston, pp. 389±476. Fohlman, J., Eaker, D., 1977. Isolation and characterization of a lethal myotoxic phospholipase from the venom of common sea snake Enhydrina schistosa causing myoglobinuria in mice. Toxicon 15, 385±393. Gopalakrishnakone, P., Chen, S.Z.J., Gwee, M.C.E., 1993. Ultrastructural changes induced by the myoglobinuria-producing venom from Pseudechis australis in mice kidney. In: Tan, N.H., Oo, S.L., Thambyrajah, V., Azila, N. (Eds.), Advances in Venom and Toxin Research: Animal, Plant, and Microbial Toxins. Malaysian Society on Toxinology, Kuala Lumpur, pp. 129±134. Gopalakrishnakone, P., 1997. Myotoxic phospholipases from snake venoms: general myoglobinuria and local myonecrotic toxins. Toxicon 35, 476. GutieÂrrez, J.M., Lomonte, B., 1995. Phospholipases A2 myotoxins from Bothrops snake venoms: Review article. Toxicon 33, 1405±1424. GutieÂrrez, J.M., Chaves, F., Gene, J.A., Lomonte, B., Camacho, Z., Schosinsky, K., 1989. Myonecrosis induced by a basic myotoxin isolated from the venom of the snake Bothrops asper. Exp. Molec. Path. 52, 25±36. GutieÂrrez, J.M., Rojas, G., Jorge de Silva Jr, N., Nunez, J., 1992. Experimental myonecrosis induced by the venoms of South American Micrurus (Coral snake). Toxicon 30, 1299±1302. Harris, J.B., 1991. Phospholipases in snake venoms and their eects on nerve and muscle. In: Harvey, A.L. (Ed.), Snake Toxins. Pergamon Press, New York, pp. 91±129. Huang, M-Z., Wang, Q.C., Liu, G.F., 1993. Eects of an acidic phospholipase A2 puri®ed from Ophiophagus hannah (king cobra) venom on rat heart. Toxicon 31, 627±635. Huang, M-Z., Gopalakrishnakone, P., 1996. Pathalogical changes induced by an acidic phospholipase A2 from Ophiophagus hannah venom on the heart and skeletal muscle of mice after systemic injection. Toxicon 34, 201±211. Iat, F., 1988. Marine snakes of Pakistan. Rec. Zool. Surv. Pak. 11, 1±56. Johnson, E.K., Ownby, C.L., 1993. Isolation of a myotoxin from the venom of Agkistrodon contortrix laticinctus (broad-banded copper head) and pathogenesis of myonecrosis induced it in mice. Toxicon 31, 245±255. Kaplowitz, N., 1992. Drug metabolism and hepatotoxicity. In: Kaplowitz, N. (Ed.), Liver and Billary Diseases. Williams & Wilkins, Baltimore, pp. 82±97. Kini, R.M., Iwanga, S., 1986. Structure-function relationships of phospholipases I: Prediction of presynaptic neurotoxicity. Toxicon 24, 527±541. Kini, R.M., Evans, H.J., 1989. A model to explain the pharmacological eects of snake venom phospholipases A2. Toxicon 27, 613±635. Kini, R.M., Evans, H.J., 1990. Eects of snake venom proteins on blood platelets. Toxicon 28, 1387± 1422.
704
S.A. Ali et al. / Toxicon 38 (2000) 687±705
Lomonte, B., GutieÂrrez, J.M., 1989. A new muscle damaging toxin, myotoxin II, from the venom of the snake Bothrops asper (terciopelo). Toxicon 27, 725±733. Mancuso, L.C., Correa, M.M., Vieira, C.A., Cunha, O.A.B., Lachat, J-J., Selistre de Araujo, H.S., Ownby, C.L., Giglio, J.R., 1995. Fractionation of Bothrops pirajai snake venom: isolation and characterization of piratoxin-I, a new myotoxic protein. Toxicon 33, 615±626. Marsden, A.T.H., Reid, H.A., 1961. Pathology of sea snake poisoning. Brit. Med. J. 1, 1290±1293. Mebs, D., Samejima, Y., 1986. Isolation and characterization of myotoxic phospholipase A2 from crotalid venoms. Toxicon 24, 161±168. Mebs, D., Ownby, C.L., 1990. Myotoxic components of snake venoms: their biochemical and biological activities. Pharmac. Ther. 48, 223±236. Minton Jr, S.A., 1974. Snake and snake venoms. In: Kugelmass, N. (Ed.), Venom Diseases. Thomas, Illinois, pp. 107±124. Nakai, M., Nakashima, K.I., Ogawa, T., Shimohigashi, Y., Hattori, S., Chang, C.C., Ohno, M., 1995. Puri®cation and primary structure of a myotoxic lysine-49 phospholipase A2 with low lipolytic activity from Trimeresurus gramineus venom. Toxicon 33, 1469±1478. Onrat, D., Pakmanee, N., Thammapalert, W., Tampitag, S., 1993. Characterization of local tissue necrotic factor(s) fraction I and XIII from Siamese Cobra venom. In: Tan, N.H., Oo, S.L., Thambyrajah, V., Azila, N. (Eds.), Advances in Venom and Toxin Research: Animal, Plant and Microbial Toxins. Malaysian Society on Toxinology, Kuala Lumpur, pp. 141±146. Ouyang, C., Teng, C.M., Huang, T.F., 1987. Characterization of snake venom principles aecting blood coagulation and platelet aggregation. Asia Paci®c J. Pharm. 2, 169±179. Ownby, C.L., Collerg, T.R., White, S.P., 1997. Isolation, characterization and crystallization of a phospholipases A2 myotoxin from the venom of the prairie rattle snake (Crotalus viridis viridis ). Toxicon 35, 111±124. Ponraj, D., Gopalakrishnakone, P., 1995. Morphological changes induced by a generalized myotoxin (myoglobinuria-inducing toxin) from the venom of Pseudechis australis (king brown snake) in skeletal muscle and kidney of mice. Toxicon 33, 1453±1467. Preston, S.A., Davis, C.E., Harris, J.B., 1990. The assessment of muscel ®bre loss after the injection of the venom of Notechis scutatus (Australian Tiger Snake). Toxicon 28, 201±214. Reid, H.A., 1961. Myoglobinuria and sea snake bite poisoning. Brit. Med. J. 1, 1284±1289. Rosenberg, P.R., 1986. The relationship between enzymatic activity and pharmacological properties of phospholipases in natural poisons. In: Harris, J.B. (Ed.), Natural Toxins. Clarendon Press, Oxford, pp. 129±174. Selistre, H.S., Queiroz, L.S., Cunha, O.A.B., De Souza, G.E.P., Giglio, J.R., 1990. Isolation and characterization of hemorrhagic, myonecrotic and edema-inducing toxins from Bothrops insularis (jararaca ilhoa) venom. Toxicon 28, 261±273. Soares, A.M., Rodrigues, V.M., Homsi-Brandeburgo, M.I., Toyama, M.H., Lombardi, F.R., Arni, R.K., Giglio, J.R., 1998. A rapid procedure for the isolation of lys-49 myotoxin II from Bothrops moojeni (Caissaca) venom: biochemical characterization, crystallization, myotoxic and edematogenic activity. Toxicon 36, 503±514. Soe-Soe, Than-Than, Khin-Ei-Han, 1990. The nephrotoxic action of Russell's viper (Vipera russelli ) venom. Toxicon 28, 461±467. Stocker, K.F., 1990. Composition of snake venoms. In: Stocker, K.F. (Ed.), Medical Use of Snake Venom Proteins. CRC Press, Boca Raton, FL, pp. 33±56. Tan, N-H., Ponnudurai, G.A., 1991. Comparative study of the biological properties of some sea snake venoms. Comp. Biochem. Physiol. 99, 351±354. Terao, K., Ohmori, S., Igarashi, K., Ohtani, I., Watanabe, M.F., Harada, K.I., Ito, E., Watanabe, M., 1994. Electron microscopic studies on experimental poisoning in mice induced by cylindrospermopsin isolated from blue-green alga Umezakia natans. Toxicon 32, 833±843. Tsai, I-H., Lu, P-J., Wang, Y-M., Ho, C-L., Liaw, L-L., 1995. Molecular cloning and characterization of a neurotoxic phospholipase A2 from the venom of Taiwan habu (Trimeresurus mucrosquamatus ). Biochem. J. 311, 895±900. Tu, A.T., 1987. Biotoxicology of sea snake venoms. Annal. Emerg. Med. 16, 149±154. Weinstein, S.A., Bell, R.C., Brown II, C.R., Fletcher, J.E., Smith, L.A., 1992. Pathologic changes
S.A. Ali et al. / Toxicon 38 (2000) 687±705
705
induced by phospholipase A2 isolated from the venom of Collett's snake, Pseudechis colletti: a light and electron microscopic study. Toxicon 30, 171±185. Yang, T.Y., Lee, C.Y., 1978. Pharmacological studies on the venom of a sea snake Hydrophis cyanocinctus. In: Rosenberg, P. (Ed.), Toxins, Animals, Plant and Microbial. Pergamon Press, New York, pp. 261±292. Zimmerman, S.E., Heatwole, H.H., Andreopoulos, P.C., Yong, L.C.J., 1992. Proliferative glomerulonephritis in mice induced by sea snake (Aipysurus laevis ) venom. Exp. Toxicol. Pathol. 44, 294±300.