Sea snake Hydrophis cyanocinctus venom. II. Histopathological changes, induced by a myotoxic...

Sea snake Hydrophis cyanocinctus venom. II.Histopathological changes, induced by amyotoxic phospholipase A2 (PLA2-H1)

Syed Abid Alia, b,*, Junaid M. Alamc, Atiya Abbasia, ZafarH. Zaidia, Stanka Stoevab, Wolfgang Voelterb

aInternational Centre for Chemical Sciences, HEJ Research Institute of Chemistry, University of Karachi,

Karachi, 75270, PakistanbAbteilung fuÈr Physikalische Biochemie, Physiologisch-chemisches Institut der UniversitaÈt, TuÈbingen,

Hoppe-Seyler-Straûe 4, D-72076, TuÈbingen, GermanycDepartment 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 andhistopathological changes in albino rats and mice. Induction of myonecrosis wasdemonstrated by their ability to release creatine kinase (CK) from damaged muscle ®bers

and direct histopathological examination of the injected muscles (i.m.). PLA2-H1 exhibitsintense myonecrosis characterized by the changes including, necrosis and edematousappearance with cellular in®ltrate, vacuolation and degenerated muscle cells with deltalesions 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 theexperimental animals and tissue samples were taken at several time intervals. Light

0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.

PII: S0041 -0101 (99)00184 -1

Toxicon 38 (2000) 687±705

www.elsevier.com/locate/toxicon

* Correspondening author. Present address: Abteilung fuÈ r Physikalische Biochemie des Physiologisch-

chemischen, Instituts der UniversitaÈ t TuÈ bingen, Hoppe-Seyler-Straûe 4, D-72076 TuÈ bingen, Germany.

Tel.: +49-07071-2975336; fax: 07071-293348.

E-mail address: saali66@hotmail.com (S.A. Ali).

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±polyacryl-

amide gel electrophoresis; MALDI MS, matrix-assisted laser desorption ionization mass spectrometery;

RP HPLC, reverse phase high performance liquid chromatography; CK, creatine kinase.

microscopic examination of the kidney sections revealed severe damage, evident by focaltubular 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 showsfatty in®ltration in parenchyma and squashed hepatocytes, while after 48 h, fattyvacuolation 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 markedin®ltration of in¯ammatory cells within alveoli. Our results suggest that the puri®ed PLA2-H1 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), skeletalmuscle damage (Preston et al., 1990; Chen et al., 1993), pneumonitis and hepatitis(Alam et al., 1995) are the few common features resulting from accidental andexperimental snake envenoming (Marsden and Reid, 1961; Reid, 1961). Thesepathological conditions have been ascribed to the e�ects of necrotic (Onrat et al.,1993) and cytotoxic components (Selistre et al., 1990; Ponraj andGopalakrishnakone, 1995; Gopalakrishnakone, 1997) in snake venom (short/longchain neurotoxins, cyto/cardiotoxins, necrotic and hemorrhagic factors/toxins andseveral enzymes), including important and the major class of enzyme, thephospholipases (Nakai et al., 1995; Huang and Gopalakrishnakone, 1996; forreview 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 toinhibit adhesion and aggregation (Ouyang et al., 1987; Kini and Evans, 1990), redcells 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; Dõ az et al., 1992; Gutie rrez and Lomonte, 1995),nerve cells to prevent neurotransmission and cause cell damage in general (Kiniand Iwanga, 1986; Harris, 1991; Chen et al., 1994; Tsai et al., 1995). Despite theirwide variety of pathophysiological/pharmacological e�ects, either dependent orindependent of their catalytic activity (Rosenberg, 1986; Kini and Evans, 1989),PLA2(s) from snake venoms show close structural similarities and constitute alarge family of a homologous (14-kDa) class of proteins (for review see Arni andWard, 1996).

Sea snakes are classi®ed in the family Hydrophiinae and Laticaudinae and arewidely distributed in the tropical Paci®c and Indian ocean. In the coastal water ofthe Pakistan region, including Karachi and Makran coasts, thirteen species of seasnakes have been reported (I�at, 1988) of which the six major species, including

S.A. Ali et al. / Toxicon 38 (2000) 687±705688

Hydrophis cyanocinctus, belong to a single genus, Hydrophis. Sea snake poisoningis a rare, but potentially serious hazard of the marine environment, as the venomscontain potent toxins and also phospholipases (Tan and Ponnudurai, 1991) beingmore lethal than venoms of many terrestrial snakes (Tu, 1987; Ali et al., 1998).Envenomations by sea snakes, although being generally considered as non-fataland 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 myotoxicphospholipase A2 (PLA2-H1), isolated from the sea snake Hydrophis cyanocinctusvenom. Associated acute nephritis, hepatitis and mild pneumonitis were alsoapparent in all test groups of animals. The present communication describes thehistopathological changes induced by PLA2-H1 in di�erent organs, pathogenesis,and possible mechanisms of such myonecrotic, nephrotic and hepatic syndromeshave 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 waterof Karachi coast (Pakistan). Snakes were identi®ed by the Zoological SurveyDepartment, 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 itschemical and biological properties was the subject of separate communicationsand described elsewhere (Ali et al., 1998, 1999). Brie¯y, puri®cation of a toxicPLA2 component (H1) from sea snake Hydrophis cyanocinctus venom wasachieved by single step RP-HPLC on a Nucleosil 7C18 column. A total of 25fractions were separated, three of which had phospholipase A2 activity. Puri®edPLA2-H1 is a 13588 Da single polypeptide chain (as determined by SDS±PAGEand MALDI MS), enzymatically active and a toxic component of the wholevenom 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 (Aliet al., 1999).

2.2. Animals

Fifty albino rats, weighing 100±150 g, of either sex and eighty Male SwissWistar mice (approximately weight 30 g) were used. The animals were housed ®veper 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.

S.A. Ali et al. / Toxicon 38 (2000) 687±705 689

2.3. Assay for generalized histopathological changes

The rats were divided into ®ve groups (I±V). Group I (10 rats) givenphysiological saline (i.p.) serve as control. Group II and III (10 rats each) wereinjected (i.p.) with 3.0 mg of puri®ed PLA2-H1 in 100 ml saline and sacri®ced after24 and 48 h, respectively. Group IV and V (10 rats each) received 6.0 mg of PLA2-H1 in 100 ml saline (i.p.) and were sacri®ced at 24 and 48 h, respectively. Micewere divided in three groups (VI±VIII, n = 6 each). Mice in groups VI and VIIreceived 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 macroscopicallyexamined and ®xed in 7% formalin. Liver, kidneys, lungs and spleen wereembedded in para�n and sliced. The slides for light microscopy were stained withhematoxylin 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 trichromestains. All preparations were made and analyzed as described earlier (Alam andQasim, 1997; Alam et al., 1993). Kidney sections from rat and mice were alsostained with Massons's trichrome stain to monitor myoglobin casts (Ponraj andGopalakrishnakone, 1995). Blood was also collected from group I through VIIIand 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 andmyoglobinuria

2.5.1. Enzyme assayThe liberation of creatine kinase from damaged muscle cells was followed by

using the enzymatic UV method (Deutsche Gesellschaft fuÈ r Klinische Chemie) asdescribed in the assay manual (Boehringer-Mannheim, Germany) to measureenzyme activity in plasma. Male Swiss Wistar mice (approximately 30 g) wereinjected in the right thigh (gastrocnemius muscle) with 1.5 mg of PLA2-H1 in 25 ml0.9% sodium chloride (w/v). The PLA2-H1 injected mice were sacri®ced at varioustime 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 abdominalcava and the plasma separated by centrifugation at 15,000 rpm for 3 min at roomtemperature. Control animals (n = 6) received 0.9% sodium chloride only(Mancuso et al., 1995; Ponraj and Gopalakrishnakone, 1995).

S.A. Ali et al. / Toxicon 38 (2000) 687±705690

2.5.2. Assay for myonecrosisFor 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 andcontrol) and post-®xed overnight in 10% formalin, dehydrated through a series ofethanol treatments and embedded in wax. Sections of 5 mm thickness were cut(Shandon AS 325, USA), stained with H & E and examined under the lightmicroscope (Nikon, Japan). Areas exhibiting pathology were photographed with aKonica (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 stainedwith H & E.

2.5.3. Assay for myoglobinuriaThe 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 andGopalakrishnakone, 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-foldincrease in toxicity over the crude venom (LD50 i.p. 0.805 mg/kg in rats and0.730 mg/kg in mice).

3.2. Generalized pathological ®ndings

After i.p. injection, PLA2-H1 was highly toxic to animals causing reducedmobility within a few hours. Generalized paralysis appeared after 10±12 h in ratsand 5±6 h in mice. Most of the animals did not survive after 35 h in all testgroups, except in groups II and III, where 4 and 3 out of 10 rats survived up to40 h, respectively. Macroscopic examination of autopsied organs in, both, rats andmice showed congested lungs, livers and swollen kidneys. Changes in the wetweight of a�ected kidneys were also noticed which was slightly higher ascompared to the control group. In addition, kidneys were tender on touching.Likewise, slightly swollen and discoloured liver was noted, specially from thoseanimals surviving upto 40 h. No visible change was observed in lungs, spleens andhearts. Notably, hearts and spleens showed no pathological changes in any group.Intramuscular injection in mice also did not induce any notable change in allorgans except the area of muscle injected. Morphologically, mild changes wereonly seen in kidneys of groups sacri®ced after 48 h.

Light microscopic examination of kidney sections, from all test groups showed

S.A. Ali et al. / Toxicon 38 (2000) 687±705 691

generalized tubular degeneration (Fig. 1B, C). However, tubular degeneration wasmore prominent in groups surviving up to 40 h, specially exhibiting a profounddegenerated epithelial lining of tubules. Complete disquamation of epithelial liningwas also noted with sporadic focal tubular necrosis (Fig. 1B, C). Markednephrotoxicity in the present study was possibly due to the fact that the kidneysare the main ®ltration organs of the blood and thus the main source ofelimination of toxaemia which is experimentally induced by PLA2 administration.Kidney sections, stained with Masson's trichrome stain, do not reveal the presenceof any myoglobin casts, but do exhibit similar pathological ®ndings in bothanimal groups, i.e. mice and rats.

Microscopic examination of liver and lungs showed considerable changes inanimals surviving upto 40 h. Marked fatty changes in liver were noted in allsubjects (Fig. 2B, C). Hepatic injury is also characterized by degeneratedhepatocytes and identi®ed by swollen edematous hepatocytes with clumpedcytoplasm and large clear spaces (Fig. 2C). Furthermore, lung sections showedin®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.

S.A. Ali et al. / Toxicon 38 (2000) 687±705692

Fig. 1 (continued)

S.A. Ali et al. / Toxicon 38 (2000) 687±705 693

However, analysis of plasma for creatine kinase liberation by lysed tissues andcells revealed a several-fold increase in its level, both, at 24 and 48 h in all groupsII through to VII (data not shown).

3.3. Myonecrosis

The maximum peak of CK liberation from damaged muscle, induced by PLA2-H1 in mice by intramuscular injection (i.m.) in groups A to I, was obtained atdi�erent time intervals. PLA2-H1-induced gradual increase in CK liberation within24 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 CKlevel gradually normalized (Fig. 4). The muscle tissue from the control group wasnormal (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. Thechanges include necrosis and edematous appearance with cellular in®ltrate (Fig.5B), vacuolation and degenerated muscle cells with delta lesions and heavy edemain between the cells (Fig. 5C, D). None of the tissue of the animals in groups A toC (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.

S.A. Ali et al. / Toxicon 38 (2000) 687±705694

Fig. 2 (continued)

S.A. Ali et al. / Toxicon 38 (2000) 687±705 695

Fig. 3. (A) Light micrograph of lung from control group receiving 100 ml saline (i.p.). Section shows

normal bronchus (arrowhead) and intact alveoli (arrow). (B) Photomicrograph of test group V

receiving 6.0 mg PLA2-H1 (i.p.) and sacri®ced after 48 h. Section shows dilated bronchus (arrowhead)

and marked in®ltration of in¯ammatory cells within alveoli (arrows) magni®cation 350 �H & E stain.

S.A. Ali et al. / Toxicon 38 (2000) 687±705696

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, spleenand heart in any of the groups examined. However, the kidney sections of thegroups sacri®ced at 48 and 72 h (groups G and H, respectively) showed mildtubular degeneration. Furthermore, fatty in®ltration in liver was observed, butonly in animals of the group sacri®ced after 24 h, probably due to the lipolyticaction of PLA2-H1. No myoglobinuria was noted in any of the groups of animalsduring the whole period of experimentation.

4. Discussion

We have examined the generalized histopathological and myonecrotic activitiesof a puri®ed PLA2-H1 from the sea snake Hydrophis cyanocinctus venom in ratsand 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 non-toxic 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) lessthan 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 snakevenom components. The results of our studies indicate that PLA2-H1 causespathological changes to kidneys, livers and lungs after i.p. injection, whereasadditionally myonecrosis was induced with mild nephritis and hepatitis after i.m.injection. Interestingly, no myonecrosis was noted in groups injectedintraperitoneally with PLA2-H1. Likewise, no myoglobinuria was detected in any

Fig. 4. Myotoxic activity of phospholipase A2 (PLA2-H1) in mice after intramuscular (i.m.)

administration followed in terms of plasma creatine kinase liberation.

S.A. Ali et al. / Toxicon 38 (2000) 687±705 697

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) A�ected muscle cells containing vacuoles (arrows),

degenerated muscle cells (double arrowheads) and marked edema (arrowhead). (D) A�ected muscle

cells with vacuoles (arrowhead) with few delta lesions (arrows) and edema (double arrowheads).

S.A. Ali et al. / Toxicon 38 (2000) 687±705698

Fig. 5 (continued)

S.A. Ali et al. / Toxicon 38 (2000) 687±705 699

of the animal groups. Masson's trichrome staining of kidneys also did not revealany myoglobin cast in kidney sections examined. However, induced nephrotoxicitywas possibly due to the fact that the kidneys are the main ®ltration organ ofblood and thus the main source of elimination of toxaemia. During theelimination of PLA2-H1 by kidney compartments (viz, tubules and glomerulus)which depend on blood supply, PLA2-H1 caused tubular and interstitial disordersthrough its cytotoxic potencies.

We have noticed marked tubular degeneration and necrosis in our study, asdescribed in the preceding section, having a generalized feature and possiblycaused by a generalized origin. However, various types of nephritis werecharacterized by several basic tissue reactions, of which hypercellularity orin¯ammatory disease are more common (Cotran et al., 1994). The in¯ammatorydisease is characterized by cellular proliferation of mesangial and endothelial cellsand leukocytic in®ltration. We have observed mild hypercellularity of lymphocyticorigin 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, whichcause vasodilation, endothelial damage and cytotoxicity. Other mechanisms mayalso be responsible in contributing towards nephrotic injury, e.g. injury ofepithelial cells, renal ablation, glomerulopathy or interstitial in¯ammation. Ofthese four, epithelial cell injury is often the result from antibody±antigen reactions(Emancipator, 1992), toxins (Ashkenazi, 1993) or/and cytokines (Border andNobel, 1993). Previously, necrotic debris and degenerated tubular epithelium wasalso noted in kidneys of mice injected with myotoxic PLA2 (Ponraj andGopalakrishnakone, 1995). Crude snake venoms also induce tubular degenerationas reported for rabbits by the injection of Vipera russelli's venom (Soe-Soe et al.,1990). Acute renal tubular degeneration and proliferative glomerulonephritis wasalso noticed in mice by the venom of the sea snake Apiysurus laevis (Zimmermanet al., 1992). Terao et al. (1994) also reported morphological changes in kidneytubules 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) onsea snake envenomation victims revealed renal damage, specially tubular necrosis,edematous interstitial necrosis and, both, di�use and focal cellular in®ltration.

Regarding the basis of hepatic injury as seen in our study (Fig. 2B, C), severalreactions have been documented preceding the hepatic injury regardless of thenature of the causative agents (Cotran et al., 1994). These reactions are necrosis,degeneration, in¯ammation, regeneration and ®brosis. In necrosis, which ismediated by toxic agents, isolated hepatocytes round up to form shrunken,pyknotic and intensely eosinolytic councilman bodies. We have noted sporadichydropic degeneration, where hepatocytes osmotically swell and rupture (Fig. 2C).Centrilobular necrosis, observed in few slides (though not very frequent), havealso been reported as a characteristic injury and occur due to many drugs andtoxins (Kaplowitz, 1992). Previously, we have also noticed similar e�ects in guineapigs induced by Hydrophis cyanocinctus whole venom (Alam and Qasim, 1993;

S.A. Ali et al. / Toxicon 38 (2000) 687±705700

Alam et al., 1995), eventually caused by the presence of some other potentnonenzymatic toxins, e.g. neurotoxins and cyto/cardiotoxins (unpublished data).Furthermore, earlier reports have also shown centrilobular degeneration andcellular in®ltration of portal areas in the liver, suggested to be due to the directe�ect of the whole venom on the hepatic system (Marsden and Reid, 1961).

In the present studies lung sections showed in®ltration of in¯ammatory cellswithin bronchus alveoli and dilation of bronchus (Fig. 3B). However, noconclusive mechanism was reported yet for pneumonitis occurring due to seasnake envenomation, even though edema and in¯ammatory changes in bronchiwas noted in the early 60 s by Marsden and Reid (1961). Therefore, pneumonitisobserved in the present studies was not due to the direct action of the venom orits components, but probably due to factors related to renal dysfunction whichinduces bronchopneumonia and acute and chronic in¯ammatory response tobroncospasm.

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 thedirect 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 andthe intensity of myonecrosis. Maximum CK liberation peaks were observed att = 12 h, 18 h and 24 h. Marked release of CK was also noted at t = 6 h and48 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 GenusMicrururus, was determined at t= 3 h and t= 6 h (Barros et al., 1994). In thepresent study it was found that the maximum peak of CK liberation did not occuralways at such a short span of time as observed with the venoms from the snakesof the Genus Micrururus. It is indicated that the occurrence of the CK liberationpeaks most likely correspond to the direct myotoxic e�ects of PLA2 in the body(Barros et al., 1994).

The type of myonecrosis that we have observed, closely relates to thosedescribed earlier by several myotoxic PLA2s (Fohlman and Eaker, 1977; Arroyo etal., 1987; Johnson and Ownby, 1993; Ponraj and Gopalakrishnakone, 1995;Huang and Gopalakrishnakone, 1996; Ownby et al., 1997) and also myotoxinsdevoid of detectable phospholipase activity (Gutie rrez et al., 1989; Gutie rrez et al.,1992; Lomonte and Gutie rrez, 1989; Johnson and Ownby, 1993; Mancuso et al.,1995; Soares et al., 1998). However, PLA2-H1 is a relatively slow acting myotoxinthan those reported earlier, indicating variations in the myotoxic potencies ofdi�erent phospholipases. Previously, Huang and Gopalakrishnakone (1996)reported on di�erences in the potencies of myotoxins, although havinghomologous structures. For example the PLA2(s) from crotalid venom producesmyonecrotic changes in muscles, but the minimum dose to induce morphologicalchanges may vary. Similarly, Trimeresurus ¯avovidis and Bothrops asper myotoxinsare about 5±10 times more active than those from Agkistrodon venoms (Fohlmanand Eaker, 1977; Mebs and Samejima, 1986).

The absence of any myotoxic activity of PLA2-H1 in lungs, hearts and spleens

S.A. Ali et al. / Toxicon 38 (2000) 687±705 701

might be caused by the non-susceptibility of the muscle and tissue towards theenzyme. This is well supported from another PLA2 from the sea snake Enhydrinaschistosa which is a highly active myotoxin, but shows no e�ect on cardiac andintrafusal muscles (Brooks et al., 1987; Huang and Gopalakroshnakone, 1996). Incontrast, PLA2 from Pseudechis colletti produces pathological changes inmyocardium and skeletal muscle (Weinstein et al., 1992). Thus, from theseinvestigations the conclusions must be drawn that di�erent PLA2(s) cause speci®cactions on di�erent 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 ofHistopathology, Liaquat National Hospital, Karachi) for her encouragement andadvice of the present study. Financial aid by Fonds der Chemischen Industrie isalso greatly acknowledged.

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