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(This is a sample cover image for this issue. The actual cover is not yet available at this time.)
This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies areencouraged to visit:
Myotoxicity and nephrotoxicity by Micrurus venoms in experimentalenvenomation
Adolfo Rafael de Roodt a,b,*, Néstor Rubén Lago a, Roberto Pablo Stock c
a Laboratorio de Toxinopatología, Centro de Patología Experimental y Aplicada, Facultad de Medicina, Universidad de Buenos Aires, Uriburu 950,5� Piso (CP C1114AAD), CABA, ArgentinabÁrea Investigación y Desarrollo, Insituto Nacional de Producción de Biológicos, ANLIS “Dr. Carlos G. Malbrán”, Ministerio de Salud, Argentinac Instituto de Biotecnología, Universidad Autónoma de México, Cuernavaca, Morelos, Mexico
a r t i c l e i n f o
Article history:Received 28 September 2011Received in revised form 9 November 2011Accepted 10 November 2011Available online 22 November 2011
Micrurus venoms are essentially neurotoxic but other activities, such as myotoxicity, maybe apparent under experimental conditions. Although this myotoxicity has been occa-sionally reported, there are no studies addressing it systematically across the genus,particularly in its relationship to other systemic manifestations such as renal impairment.The lethal potency of Micrurus fulvius, Micrurus nigrocinctus, Micrurus surinamensis,Micrurus altirostris, Micrurus balyocoriphus andMicrurus pyrrhocryptus venoms determinedby us were in the range described for the genus and all venoms exhibited phospholipaseactivity, albeit at significantly different levels. Intramuscular venom injection causedvariable local inflammation-edema; myotoxicity (as determined by plasma creatine kinaselevels and histopathology) was apparent only in those venoms with highest phospholipaseactivity, namely M. fulvius, M. nigrocinctus and M. pyrrhocryptus. Kidneys of animalsinjected with these strongly myotoxic venoms showed lesions consisting in extensivetubular necrosis with nuclear fragmentation, destruction of the brush border, rupture ofbasal membrane and epithelial exfoliation of tubular cells, granular cast and thickening oftubules. The histological characteristics of the lesions suggest an important role for indirectglomerular damage by myoglobin deposits. Phospholipase and myotoxic activities did notcorrelate significantly to the lethal potency; renal lesions were, however, evident only inthose venoms that caused extensive muscular damage. Although kidney lesions have notbeen described in clinical cases of Micrurus envenomation, the potential for nephrotoxicityof some of these venoms should be considered in the overall toxicological picture, at leastin experimental conditions.
� 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Coral snakes are the American members of the familyElapidae. Their name derives from the bright red bands ina context of black, white or yellow bands that most speciesexhibit along their body. Coral snakes are represented by
the genera Micrurus, Leptomicrurus and Micruroides, whichtogether comprise a taxonomic group of more than 120species and subspecies. Micrurus snakes (around 70species) can be found from Patagonia to North America andare responsible for envenomations throughout the Amer-icas (Campbell and Lamar, 2004).
Bites byMicrurus (M.) constitute medical emergencies inwhich the risk of death is recognized (Ministerio de Saúde,1999; Russell et al., 1997; Gold et al., 2004; Ministerio deSalud, 2007) The venom of Micrurus snakes causes loss ofmuscle strength and respiratory paralysis of peripheralorigin in animals and humans. The neurotoxic signs caused
* Corresponding author. Laboratorio de Toxinopatología, Centro dePatología Experimental y Aplicada, Facultad de Medicina, Universidad deBuenos Aires. Uriburu 950, 5� Piso (CP C1114AAD), CABA, Argentina. Tel.:þ54 9 114147 4644; fax: þ54 11 4508 3602.
0041-0101/$ – see front matter � 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.toxicon.2011.11.009
Toxicon 59 (2012) 356–364
Author's personal copy
by these venoms are the result of postsynaptic action at thelevel of end-plate receptors (a-neurotoxins, e.g. Micrurusfrontalis) and/or inhibition of evoked acetylcholine releaseby the motor nerve endings (presynaptic PLA2; b-neuro-toxins, e.g. Micrurus corallinus), and/or by depolarization ofmuscle fiber membranes (myotoxic phospholipases A2,possibly other cytotoxins, e.g. Micrurus nigrocinctus andMicrurus fulvius; Vital Brazil, 1987, 1990.) In addition,cardiovascular effects ofM. fulvius (Weis andMcIsaac,1971),and hemorrhagic effects of M. fulvius (Tan and Ponnudurai,1992), Micrurus averyi (Barros et al., 1994) and M. frontalisfrontalis (Francis et al., 1997) venoms have been described.Coagulant activity on plasma or fibrinogenwould be absentin Micrurus venoms (Barros et al., 1994.)
On the clinical side, local lesions are often minimal inMicrurus envenomations. In experimental studies, however,Micrurus ibibococca, Micrurus spixii, M. averyi, Micrurus hem-prichii and Micrurus lemniscatus venoms display inflamma-tory activity (Gutiérrez et al., 1992; Barros et al., 1994;Tambourgi et al., 1994) and some Micrurus venoms havebeen shown experimentally to produce some myotoxiceffects (Arroyo et al., 1987; Alape-Girón et al., 1996; AlapéGirón, 1997; Gutiérrez et al., 1980, 1983, 1992; Barros et al.,1994; de Roodt et al., 2002; Moraes et al., 2003; MagalhaesCamargo, 2010).
Althoughmuscular lesions have been described in humanenvenomation by Micrurus laticollaris (Pettigrew and Glass,1985), M. fulvius (Kitchens and Van Mierop, 1987) and M.lemniscatus (Manock et al., 2008), myotoxicity in humansdoes not seem to be a common clinical observation and thereis no histological evidence for it (da Silva and Bucaretchi,2003; Ministerio de Salud, 2007; Ministerio de Saúde, 1999)as described in other elapid venoms. Other elapids, such asNotechis scutatus (Karlsson et al., 1972), Oxyuranus scutellatus(Fohlman et al., 1976) and several species of Pseudechis(Leonardi et al., 1979; Mebs and Samejima, 1980; Ponraj andGopalakrishnakone, 1995) produce well documented myo-toxicity in humans and animals (Heller et al., 2007).
There is evidence, both clinical and experimental, indi-cating that myotoxicity with myoglobinuria can producerenal lesions (Acott, 1988; Ali et al., 2000; Faiz et al., 2010;Fohlman and Eaker, 1977; Gao et al., 1999; Isbister et al.,2006; Leonardi et al., 1979; Lewis, 1994; Ponraj andGopalakrishnakone, 1995, 1996, 1997; Weinstein et al.,1992; Trinh et al., 2010), however, data on myoglobinuriain Micrurus envenomations are scarce (Gutiérrez et al.,1986) and data documenting nephrotoxicity for Micrurusvenoms have not been reported. We therefore conducteda systematic in vivo study of skeletal muscle alteration andits association with kidney lesions in rats injected by theintramuscular route with venom from different species ofMicrurus from South, Central and North America.
2. Material and methods
2.1. Venoms
Micrurus fulvius (Florida, USA) andM. nigrocinctus (CostaRica) venoms were supplied by the Serpentarium “LaNauyaca”, Cuernavaca, Mexico. Micrurus surinamensis(Letizia, Colombia) was from the serpentarium of Dr. Juan
Silva Haad. The venoms of Micrurus altrirrostris, Micrurusbalyocoriphus and Micrurus pyrrhocryptus were a gift of Dr.Alejandro Urs Vogt from the Centro Toxicológico de Mis-iones (Oberá, Misiones, Argentina.) All venoms were ob-tained by manual milking, vacuum dried and stored at�20 �C until use. Venoms used as controls for some assayswere Bothrops diporus from Argentina (from the Serpen-tarium of the National Institute for Production of Biologi-cals of the National Administration of Institutes andLaboratories of Health, Ministry of Health, henceforthI.N.P.B.) and Naja nigricollis (Latoxan, France).
2.2. Animals
Wistar rats (250 g) and CF-1 mice (18–22 g) were ob-tained from the I.N.P.B. Animal Facility or from GEMABiotech, Buenos Aires Argentina. Animals were kept inplastic cages receiving water and food ad libitum undercontrolled temperature conditions. Ethical guidelinesregarding the management of animals were those recom-mended by the National Research Council (2002).
2.3. Toxic and enzymatic activities
2.3.1. Lethal potencyThe lethal potency of the venoms was determined by
the method of Molinengo as modified by Meier andTheakston (1986) in CF-1 mice (18–22 g) by the intraperi-toneal (i.p.) route with venom diluted in NaCl 0.15 M ina final volume of 0.5 ml. Lethal potency was used to adjustthe challenge doses for the other in vivo toxicity assays.
2.3.2. Coagulant activitiesSince intravascular coagulation and hemodynamic
alterations can lead to renal failure, the coagulant activity ofthe different venoms on plasma and bovine fibrinogenwere determined exactly as described by Theakston andReid (1983).
2.3.3. Hemorrhagic activitySince the hemorrhagic activity of venoms can lead to
local damage and, consequently, muscular lesions, thisactivity was studied for all the venoms as described byGutiérrez et al. (1985) with some minor modifications.Briefly, different doses of venom, ranging from 6.25 up to100 mg (depending on venom lethality in mice), were usedand mice were sacrificed after 90 min; at this time all micewere alive even at the maximal challenge doses.
2.3.4. Phosholipase activitySince phospholipases have been described as one of the
principal causes of myotoxicity in elapid venoms (Helleret al., 2007), the hydrolytic activity of the venoms wasdetermined on egg yolk (de Roodt et al., 2005). Briefly,15 ml of 1% agarose (Sigma) in 0.15 M NaCl containing0.5 ml of egg yolk and 100 ml of 100 mM CaCl2 were placedin Petri dishes. After solidification, different doses of thevenoms diluted in 40 ml of 0.15 M NaCl were placed inwellspunched in the solid phase (5 mm diameter). After incu-bation at room temperature for 48 h, the haloes of hydro-lysis were measured and potency was calculated as the
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dose of venom in micrograms that produced a hydrolytichalo with a diameter of 2 cm. Experiments were performedat least in triplicate.
2.3.5. Hemolytic assaysHemolysis by the venoms was measured two ways. An
indirect assaywas used to assess phospholipase-dependenthemolytic activity. A direct hemolysis test was performedto establish whether directly hemolytic components werepresent in the venoms (Kumar et al., 1977), sincecardiotoxin-like components with direct hemolytic activityhave been described as important myotoxic agents in someelapid venoms (Mebs and Ownby, 1990) and their presencehas been suggested for someMicrurus venoms (Vital Brazil,1987.) All experiments were performed by triplicate and atleast three times.
2.3.5.1. Indirect hemolysis assay. This activity was deter-mined using the technique described by Al-Abdulla et al.(1991), with some modifications. Briefly, samples contain-ing 20 ml of human erythrocyte pellets, 20 ml egg yolk, 10 mlof 100 mM CaCl2 were brought up to a final volume of1.2 ml with a 0.01 M phosphate buffered saline solution, pH7.4 (PBS). They were subsequently incubated for 90 min atroom temperature with different venom concentrations(0.1–8.0 mg). As positive control erythrocytes were treatedwith B. diporus venom under the same conditions. Negativecontrols consisted in the addition of 100 ml of PBS withoutvenom. After incubation, the reaction was stopped byaddition of 100 ml of 0.5 M EDTA and cooling to 4 �C. Thetubes were then centrifuged (2000 g, 10 min) and theabsorbance of the supernatant at 550 nm was determinedin a spectrophotometer. Hemolytic activity was expressedas IHD50 (mean hemolytic dose), which indicates themicrograms of venom necessary to produce 50% hemolysis.This system measures both direct and indirect hemolyticeffects, i.e. total hemolytic capacity of the venom.
2.3.5.2. Direct hemolysis assay. Direct hemolysis was deter-mined as above but without providing the system with anexternal source of phospholipids and chelating calciumwith 100 mM EDTA. This system makes it possible toobserve hemolysis that is not dependent on phospholipaseactivity. As positive control we used N. nigricollis venom.The venoms of M. fulvius, M. nigrocinctus and M. pyr-rhocryptus were also tested at higher doses (8.0–20.0 mg).
2.3.6. Creatine kinase activityFollowing the recommendations of the World Health
Organization (WHO, 2010), myotoxicity was studied bydetermining the plasmatic rise of creatine kinase activityafter intramuscular injection of the venoms. This activitywas measured using the CK-NAC kit (Wiener Laboratories,Rosario, Argentina). Wistar rats (200 g, six for each venomand controls) where lightly anesthetized with ether andinjected intramuscularly (i.m.) in the tibialis anteriormuscle of the foot, with 50 mg of venom of Micrurus altir-ostris or M. surinamensis (doses over 50 mg killed the ratsbefore 6 h) or 100 mg of the other venoms. Venoms werediluted in 50 ml of 0.15 M NaCl. Rats injected in the same
muscle with 50 ml of 0.15 M NaCl were used as control. Allanimals were lightly anesthetized with ether and bled fromthe tail vein at hours 2, 6 and 24 post-injection. Values areexpressed as U/L (units/liter).
2.3.7. Measurement of uremiaTo evaluate possible alterations in renal filtration and,
consequently, renal damage, we determined the level ofplasma urea in rats injected with the different venoms.Wistar rats (6 per venom) were injected as described forCK measurement and blood samples were collected at 2and 24 h. Controls were injected with 0.15 M NaCl aspreviously described. Plasma urea was determined usinga Uremia kit (Wiener Laboratory, Rosario, Argentina.)Detection of urea is based on the measurement of blueindophenol produced by phenol and hypochlorite in basicmedium from the CO2 and NH3 produced by urease. Thereaction was read at 540 nm and values of circulating ureaare expressed in mg/dl.
2.3.8. Inflammation and edemaAs a measure of the local activity of the venoms the
inflammation-edema in injected limbs was estimated.Limbs of rats injected with venoms as described for CKactivity (n¼ 5 per venom), were excised at the coxofemoraljoint and weighed. The difference in weight of the limbinjected with venom with respect to the contralateralcontrol (injected with NaCl 0.15 M) was expressed asa percentage.
2.4. Histopathology
After 24 h of venom injection as described, rats wereanaesthetized (diethyl ether), sacrificed and the necropsywas immediately performed. Samples of muscles injectedwith venom, control limbs and kidneys of injected rats andcontrols were preserved in 10% formaldehyde. Sampleswere embedded in paraffin and stained with hematoxylin –
eosine, Periodic Acid Schiff, Prussian blue and Masson Tri-chromic stain.
2.5. Statistics
Results are expressed as means � standard deviation, oras means with their 95% confidence intervals. When neces-sary Student’s t test was used for comparisons betweengroups. For all the statistical calculations we used the soft-ware package Prism 4.0 (Graph Pad Inc., San Diego, CA).
3. Results
Lethal potencies are summarized in Table 1. The venomof M. fulvius was the only one with a slight hemorrhagicactivity at doses approaching the lethal dose, in accordancewith previous reports (Tan and Ponnudurai, 1992). None ofthe venoms showed coagulant or thrombin-like activity,although at long incubation times (>2 h) venoms fromNorth AmericanMicrurus caused a slight physical alterationof the fibrinogen solution but without clot formation.When the solutions treated with M. fulvius and M. nigro-cinctus venoms were centrifuged (2000 g, 15 min),
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a precipitate of around 10% of the total volume wasapparent; this precipitate was not observed in identicalfibrinogen solutions treated with the other venoms (datanot shown).
Phospholipase activity was detectable in all venoms byegg yolk hydrolysis. The hydrolytic doses, in decreasingorder of potency, were M. nigrocinctus, M. pyrrhocryptus,M. fulvius, M. altirostris and M. balyocoriphus and M. sur-inamensis. The activity of the last two venoms was signifi-cantly lower than the rest (p < 0.05; Table 1).
In agreement with the results obtained from the phos-pholipase assays, all venoms were indirectly hemolytic.The venom of M. surinamensis had the lowest potency,followed by the venoms of M. balyocoriphus, M. altirostris,M. pyrrhocryptus, M. nigrocinctus and M. fulvius. The venomsof M. fulvius, M. nigrocinctus and M. pyrrhocryptus hadsignificantly higher activities than the rest (p< 0.05; Table 1.)M. surinamensis venomhad the lowest hydrolytic and indirecthemolytic activities, in concordance with previous reports(Aird and da Silva, 1991; Olamendi-Portugal et al., 2008).
None of the venomswere directly hemolytic, even at thehighest doses, with values near those observed in thenegative controls. OnlyM. fulvius venomwas slightly activeat a dose of 8 mg (p¼ 0.010 and t¼ 3.014). Using the highestdose tested (20 mg), the venoms of M. nigrocinctus and M.pyrrhocryptus exhibited differences with respect to thenegative controls (p < 0.05 and t > 2.4), however at thisdose of venom the hemolytic activity was very low bycomparison to the positive control: 5- (M. fulvius), 17- (M.nigrocinctus) or 33-fold (M. pyrrhocryptus) doses werenecessary to attain the effect of N. nigricollis venom.
All venoms increased plasma CK levels, which peaked6 h after venom injection (Fig. 1). At 24 h after injection,levels above the controls were maintained in all cases(p < 0.05). The venom ofM. fulviuswas the one that causedthe greatest increase in CK at 6 h, statistically significantwith respect to those of M. altirostris, M. surinamensis andM. balyocoriphus (p < 0.05, t > 2.2) but the differences withM. nigrocinctus and M. pyrrhocryptus venoms were notsignificant (p > 0.05, t < 1.8). The difference between thelast two were not significant (p > 0.41, t ¼ 0.835). Thevenoms of M. balyocoriphus, M. altirostris and M. sur-inamensis were the least potent (p < 0.05). The results aresummarized in Table 1 (CK at 6 h post-injection) and Fig. 1
(kinetics of CK at 2, 6 and 24 h post-injection). Uremia wassignificantly increased with respect to controls after 24 honly in rats injected with the venoms of M. pyrrhocryptusand M. fulvius (p < 0.05; t > 2.6; Table 1.)
Macroscopic observation of injected limbs in almost allcases showed inflammation and edema. M. pyrrhocryptusvenom was the most inflammatory (p < 0.01, t > 5.0), fol-lowed by the venoms of M. fulvius and M. nigrocinctus; M.surinamensis venom was the least inflammatory (Table 1,Fig. 2).
Histopathological analysis revealed that muscularlesions consisted in necrosis, edema and inflammatoryinfiltrationwith disruption of muscular fibers (Fig. 3) in ratsinjected with venoms that produced the greatest increasein CK (Table 1 and Fig. 1). Analysis of muscles from ratsinjected with M. fulvius and M. nigrocinctus venomsrevealed severe myofibrillar necrosis. Rats injected with M.pyrrhocryptus venom displayed a lower degree of myo-necrosis, which was absent or very minor with theremaining venoms.
The kidneys of rats injected with M. fulvius or M.nigrocinctus venoms exhibited nuclear fragmentation,destruction of the brush border, rupture of basal membrane
Table 1Toxic and enzymatic activities of the venoms of six species of Micrurus. Lethal potency is expressed in mg/g by the i.p. route in CF-1 mice; Indirect hemolyticactivity is expressed as IHD50 in micrograms; Hydrolysis of egg yolk is indicated as the amount of venom in micrograms that produces a hydrolytic halo of2 cm; creatine kinase activity (CK) after 6 h of intramuscular inoculation is expressed in U/L; Uremia after 24 hours is expressed in mg/dl; Inflammation isexpressed as percentage of difference of weight between the limb injected with venomwith respect to the contralateral limb injected with NaCl 0.15 M, 24 hpost-injection.
Fig. 1. Plasma creatine kinase (CK) activity after intramuscular injection ofMicrurus venoms. Bars indicate the mean values (�SEM) of rats injectedwith the different Micrurus venoms (n ¼ 6) and controls injected with NaCl0.15 M at 2, 6 and 24 h post-injection. Levels above the controls weremaintained in all cases 24 h after injection (p < 0.05). The venom of M.fulvius was the one that caused the greatest increase in CK at 2 and 6 h. Theasterisk indicates that this increase was statistically significant with respectto M. altirostris, M. surinamensis and M. balyocoriphus (p < 0.05).
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and epithelial exfoliation of tubular cells, presence ofgranular cast and thickening of tubules. The presence ofabundant proteinaceous material and granular cylinders inthe tubules of different sections of the nephron is evident(Fig. 4a and c). Occasionally, glomerular congestion and thepresence of intracapillary thrombi were observed inglomeruli of rats injected with these two venoms (Fig. 4b).The Prussian blue technique revealed small granulardeposits of salts in tubules. Rats injected with M. pyr-rhocryptus venom exhibited these same lesions but toa smaller extent whereas the other venoms only producedisolated and mild renal alterations.
4. Discussion
In our laboratory we had observed macroscopic myo-globinuria and the presence of dark urine in the bladder ofmice challenged with high doses of M. nigrocinctus and M.fulvius venoms. These observations suggested that,although not reported, renal lesions could occur at least inexperimental envenomation by some species of Micrurus.We therefore set out to examine the venom of a number ofspecies of Micrurus, ranging from South to North America,in a comparative study of their toxic profiles in terms oflethality, phospholipase activity, hemolytic potency and
Fig. 2. Macroscopic inflammation and edema in limbs of rats injected with M. nigrocinctus (2a), M. fulvius (2b) and M. pyrrhocryptus (2c) venoms. Rats wereinjected in digital extensor muscles. V indicates the muscles injected with venom (100 mg in 50 ml of NaCl 0.15 M) and C muscles injected with 50 ml of NaCl0.15 M.
Fig. 3. Muscular damage in rats injected with Micrurus venoms. 3a. Muscle injected with M. fulvius venom; the deeply eosinophilc necrotic cells show nuclear lossand the extensive necrotic area (asterisk) is surrounded by injured viable myocytes, edema and inflammation (40� original magnification.) 3b. Skeletal muscleinjected with M. nigrocinctus venom, stained with hematoxilin and eosin. Mononuclear inflammation and focal necrosis (asterisk) are apparent (250� magnifi-cation.) 3c. Normal skeletal muscle. 3d. Cross-section shows intrasarcoplasmatic necrosis and disintegrating sarcolemmal structures, edema and mononuclearinfiltrate (400� magnificantion.).
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Author's personal copy
myotoxicity in order to dissect the probable causes of thisrenal impairment.
All venoms were of high lethal potency under theconditions of the study, in the range previously reported forMicrurus (Bolaños et al., 1978; Sanchez et al., 1992; Tan andPonnudurai, 1992; Higashi et al., 1995; da Silva and Aird,2001; de Roodt, 2002; de Roodt et al., 2004; Tanaka et al.,2010). When compared to other elapid venoms (Broadet al.,1979),Micrurus lethal potency values are intermediate.
All venoms, when injected intramuscularly, increaseduremia levels with respect to controls at 24 h post-injec-tion. The greatest increase was seen with the venoms of M.pyrrhocryptus and M. fulvius. Uremia elevation is a robustindicator of renal impairment (Zotta et al., 2008), sinceplasma urea is elevated over baseline values whenglomerular filtration is under 50%. The basis of kidneydysfunction was established by histological analysis, whichrevealed that M. fulvius, M. nigrocinctus and, to a lesserdegree, M. pyrrhocryptus were capable of causing impor-tant renal lesions. Kidneys from rats treated with thesevenoms showed extensive tubular necrosis of epithelialcells, with rupture of basal membrane and epithelial exfo-liation in the lumen. Tubular epithelia were thickened andthe presence of proteinaceous material and granular castincluding cylinders in tubules was most notable in ratsinjected with M. fulvius and M. nigrocinctus venoms. Castswere seen in proximal and distal tubules, loops of Henle
and in collecting ducts. In some tubules the castscompletely obstructed the lumen. Glomeruli showedcongestion and presence of intracapillary thrombi. Kidneysof rats injected with the other venoms were only mildlyaffected even at venom doses near the lethal dose.
Snake venom nephrotoxicity can have several origins,such as impairment of perfusion due to intravascular coag-ulation (Rezende et al., 1989; Burdmann et al., 1993), directaction of cytotoxic venom components on kidney structures(Chugh, 1989; Ali et al., 2000; de Castro et al., 2004; Havtet al., 2005; Evangelista et al., 2010) and hemoglobin ormyoglobin deposits in renal tubules (Azevedo-Marqueset al., 1985; Weinstein et al., 1992; Lewis, 1994; Ponraj andGopalakrishnakone, 1995, 1996, 1997; Gao et al., 1999; Aliet al., 2000; Isbister et al., 2006; Faiz et al., 2010; Trinhet al., 2010). We examined the probable causes of theevident kidney damage sequentially in terms of these knowncauses.
In the case of the Micrurus venoms studied, intravas-cular coagulation does not seem to be involved in kidneydamage as these venoms did not exhibit coagulant activityon plasma and no evidence of disseminated intravascularcoagulation was found in the histological studies.
Another possible cause of kidney impairment are myo-and nephrotoxic components of some elapid venomscardiotoxins (Mebs and Ownby, 1990). In the case of ourMicrurus venoms, we did not detect direct hemolytic
Fig. 4. Kidney damage in rats injected with Micrurus venoms. Hematoxylin and eosin staining. Presence of proteinaceous material and granular cylinders in thetubules of the different sections of the nephron (4a and 4c). Glomerular congestion and intracapillary thrombi in rats injected with M. fulvius and M. nigrocinctusvenoms (4b). Original magnification 100�. Lesions consisted in nuclear fragmentation, destruction of the brush border, rupture of basal membrane and epithelialexfoliation of tubular cells (4a and 4c). Original magnification 250�. Markers: Tubular necrosis (*), nuclear fragmentation (#), exfoliation of tubular cells ($),granular cast (þ), glomerular capillary lesion (X). Normal control (4d).
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activity which could signal the presence of cytotoxic factorssuch as hemolytic cardiotoxins (Kumar et al., 1977; Hiderand Khader, 1982; Chen et al., 1984; Hodges et al., 1987;Kini and Evans, 1989; Dufton and Hider, 1991; Jiang et al.,1989; Ma et al., 2002; Troiano et al., 2005; Kao et al.,2010). Although other techniques may be more sensitiveto detect cytotoxic/hemolytic activity, at the venom dosesused in our study the presence of cardiotoxins would havebeen detected. We could only establish a very low directhemolytic activity in M. fulvius venom, in which car-diotoxins have been described (Vital Brazil, 1987). From 3-to 6-fold, or over 17-fold, doses of the other Micrurusvenoms were necessary for a hemolytic effect comparableto that of N. nigricollis venom over the negative controls.The barely detectable direct hemolytic activity at the highdoses of venom used establishes that these venoms do notcontain, or contain very low amounts, of cardiotoxin-likecomponents and, therefore, argues against a direct cyto-toxic effect at themuscular level by this type of component.
The possible mechanism of renal damage in these casesis indirect, by way of deposits of hemoglobin or myoglobinwhich compromise renal structure and function. The lowhemolytic activity of the venoms, coupled to the doses used,argue against massive hemolysis as the cause of glomerulardamage. The extensive renal lesions were observed in ratsinjected with the venoms of highest myotoxic and phos-pholipase activity.
Although all venoms studied had detectable PLA2
activity, this activity was not directly related to lethalpotency but it correlated significantly with CK levels. Infact, venoms with highest lethal potency were those withlowest PLA2 activity (as determined by egg yolk hydrolysis[r2 < 0.47] and indirect hemolysis [r2 < 0.20]). Correlationbetween phospholipase and indirect hemolytic activitieswas, as expected, positive and significant (r2 ¼ 0.85 and thecorrelation between the CK levels and PLA2 activity wasr2 ¼ 0.64). Phospholipases have been described as one ofthe principal myotoxic components in elapid venoms(Fohlman and Eaker,1977;Mebs and Samejima,1980;Mebsand Ownby, 1990; Ali et al., 2000; Gao et al., 1999; Lee et al.,1989; Jiang et al., 1989).
Precipitation of proteins in tubules has been observed inenvenomations by some vipers (Azevedo-Marques et al.,1985) and in some experimental elapid envenomations(Mebs and Samejima, 1980), and clinically in humans (Tuand Miller, 1989; Gao et al., 1999) and domestic animals(Heller et al., 2007). Characteristic of renal lesions by myo-globinuria is the presence of cast showing mild brownpigmentation, usually of granular appearance with irregularglobules. Another characteristic is tubular damage withexfoliation of epithelial cells and thinning of the epithelium(Racusen and Kashgarian, 2007). The presence of myoglobindeposits in kidneys of rats injected withMicrurus venoms issupported by the results of 1) Prussian blue staining, whichrevealed salt deposits in renal tubules of rats injected withM. fulvius andM. nigrocinctus venoms and2) the brown colorof the cast when stained with hematoxilin-eosin and thelight red color when stained with Masson Trichromic stain(Racusen and Kashgarian, 2007).
Some elapid venoms (Leonardi et al., 1979; Tu andMiller, 1989; Ponraj and Gopalakrishnakone, 1995; Ali
et al., 2000; Isbister et al., 2006; Trinh et al., 2010; Faizet al., 2010) and viper venoms (Azevedo-Marques et al.,1985) can cause systemic myotoxicity. In the case of theMicrurus venoms used in our study, the fact that neither thecontralateral nor other striated muscles of rats injectedwith M. fulvius, M. nigrocinctus and M. pyrrhocryptusvenoms exhibited necrosis or inflammation indicates thatnecrosis caused by these venoms is strictly local.
The elevated CK levels induced by the more nephrotoxicvenoms suggest that severe muscular damage is the sourceof myoglobin. Miotoxicity with increase of CK levels hasbeen reported previously for the venom of M. nigrocinctusand its isolated phospholipases (Gutiérrez et al., 1986;Arroyo et al., 1987). The venoms of M. surinamensis, M.altirostris andM. balyocoriphus produced isolatedmild renalalterations in just a few cases. In our hands,M. surinamensisvenomwas not myotoxic (contrary to the results of Cecchiniet al., 2005), in agreement with the results of Barros et al.(1994). When injected intramuscularly, this venom onlycaused a slight increase in CK, very mild inflammation andno renal damage. In this respect M. surinamensis venomseems to differ substantially from other coral snake venomsdescribed to date (Aird and da Silva, 1991; Olamendi-Portugal et al., 2008). Myotoxicity was also low for M. altir-ostris venom, contrary to the results of Moraes et al. (2003).These discrepancies could be attributed to distinct toxicactions on different animal models, differences at the levelof the muscle used for injection (Melo and Ownby, 1996), orvariability betweenM. altirostris venoms from NortheasternArgentina and fromBrazil (Moraes et al., 2003) and betweenM. surinamensis venoms from Brazil (Cecchini et al., 2005)and Colombia (this study).
In human envenomation by some species ofMicrurus anincrease in plasmatic CK activity has been reported(Pettigrew and Glass, 1985; Kitchens and VanMierop,1987;Manock et al., 2008); histological evidence of myotoxicityis, however, lacking. Although renal lesions do not seem tobe a major consequence of human or domestic animalenvenomation by Micrurus, at least for some venoms insome experimental models the possibility of renal lesionsshould be considered in the global toxicity. This knowledgemay be if use in the diagnosis and treatment of clinicalcases in which unusual symptoms are manifest, i.e. beyondthe expected neurotoxicity of a coral snake envenomation.To our knowledge, this is the first study of renal lesions inexperimental Micrurus envenomations and on the rela-tionship between phospholipase activity and myotoxicity-nephrotoxicity, as determined by toxicological and histo-logical studies.
Acknowledgments
The authors are very grateful for the technical assistanceof Mr. Víctor Marcelo Manzanelli in the determination oftoxic activities of the venoms and for the technical assis-tance of Mr. Claudio Mirabelli in processing the samples forthe histopathological studies (GEMA Biotech Argentina).The authors thank Dr. Alejandro Urs Vogt, Director of theCentro Zootoxicológico de Misiones (Oberá, Misiones,Argentina), for donation ofMicrurus venom of species fromArgentina. We are grateful to Instituto Bioclon S.A. de C.V.
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