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TOXIC ACTIVITIES OF VENOMS FROM NINE BOTHROPS SPECIES AND THEIR CORRELATION WITH LETHALITY AND NECROSIS Moxicw

L. F>

w,l Axw

M . MOURA-DA-SILVA, 1* and I. Morwl

F. O. S. Fxwxçw,2 J. L. Cwxnoso2

LCentro de Imunologia O . G. Bier; and'Hospital Vital Brazil-Instituto Butantan, Silo Paulo, SP, Brazil (Received 26 Afay 1992 ; accepted 11 July 1992)

L. Few, A. M . MOUitw-DA-SILVA, F. O. S. FRwxçw, J. L. CARDOSO and I. Mores . Toxic activities of venoms from nine Bothrops species and their correlation with lethality and necrosis. Toxicon 30, 1603-1608, 1992 .-The comparison of seven toxic activities contained in venoms from nine different species of Bothrops and the correlation of each activity with lethality and necrosis was the subject of this study . The haemorrhagic, coagulant, necrotizing, myotoxic, proteolytic and phospholipase activities were not equally distributed among the venoms studied except for the oedema-inducing activity which was almost equally distributed among them. The correlation coef&cient was estimated for each activity in relation to lethality and necrosis induced by the venom. Lethality was significantly related to haemortagic and oedema-inducing activities, whereas the necrotizing activity showed significant correlation with phospholipase and coagulant activities . Proteolytic activity presented a significant inverse correlation with lethality. M.

INTRODUCTION

Ix Bxwz>z., the genus Bothrops contains several species and subspecies of snakes responsible for about 90% of the ophidic accidents. Bothropic accidents are characterized by haemorrhage, oedema, necrosis and alterations in the coagulant system . In cases of severe envenoming these effects can lead to complete destruction and subsequent loss of a limb or even death (RosExl~.n, 1971). These symptoms are due to the additive or synergic effect of the different toxins and enzymes present in the venoms (GvTtERxI=a and LOMONTE, 1989). Interspecific variations are well documented among venoms from snakes of the same genus (Ct~Pwux et al., 1991). In the genus Bothrops, coagulant activity (Nwxws et al., 1979), necrotizing factors (VII.LARROSI, et al., 1978/1979) as well as myotoxins (MOURADw Sa.vw et al., 1990) are not equally distributed in venoms of each species. Age and geographical variability is also known for Bothrops snakes (ARwooN and Gussxsx, 1981 ; M1:IER, 1986). In experimental models, lethality of different venoms of the Bothrops species may vary up to five-fold in Lnso values (Dews-Dw-Su,vw et al., 1989). These observations Author to whom correspondence should be addressed. 1603

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M. L. FERREIRA et a1.

indicate that the envenoming due to accidents by each species of Bothrops may differ according to the amount and quality of the different toxins present in each venom. We report here a comparative study of seven toxic activities contained in venoms from nine different species of Bothrops, attempting to correlate each activity with the lethality or the local necrosis induced by the different venoms . This approach may be important in order to predict the development of the intoxication by the different species according to the experimentally defined toxic activities present in their venoms . Venons

MATERIAL AND METHODS

Desiccated venoms of B. alternates, B . atrox, B. erythromelas, B. cohere, B. jararaca, B. jararacwsse, B. moojeni, B. neewiedi and B. pradoi were supplied by Instituto Butantan (Sâo Paulo, Brazil) . Tlvrty to 40

snakes of each species were milked and the venoms pooled. Their ~w corresponded to 66 pg/mouse (B. alternator), 145 pg/mouse (B. atrox), 46 .7 pg/mouse (B. cohere), 7011g/mouse (B. erythromtlas), 3311g/mouse (B . jararaca), 7611g/mouse (B. jararacessu), 115.2 pg/mouse (B. moojenl), 35 .511g/mouse (B. neuwiedi) and 61 .51ig/mouse (B. prado~ (Des-D~-Sn.ve et al., 1989). Animals

Outbred mice, male, weighing 18-22 g, supplied by the animal house of Instituto Butantan (Sâo Paulo, Brazil), were used throughout . Estimation of Iraemorrhggic activity

Haemorrhagic activity was estimated according to Kormo et a1. (1960) with the modifications introduced by Owerev et a1. (1984). Briefly, mice were shaved in the backs and injected i.d . with 50111 of the solutions

containing 10118 of each venom in saline . After 2hr, the mice were killed with ether, the skin stripped off from the dorsum and placed on a plank. Two diameters were determined for the haemorrhagic spot by measuring the longest diameter and We one perpendicular to the longest. Results are expressed as the product of the diameters multiplied by a factor varying from 1 to 4 according to the intensity of the colour of the spot (mm= /118 of venom). Estimation ojnecrotizing activity

Necrosis was quantitated after an i.d . injection of 100118 of each venom contained in 501d of saline into the shaved backs oC the mice . After 72 hr, the animals were killed with ether, and the skin removed. The necrotic area was measured as described for the haemorrhagic activity. In this case, the factor from 1 to 4 corresponded to the profundity of the lesion in the akin . Results are expressed in mm=/118 of venom. Estinurtion of oedema-indeclng activity

The oedema-inducing activity of the venoms was assayed according to the method of Y~utuwe et al. (1976). Mice were injected in the foot pad of the hind limb with 50 pg of each venom in 101d of saline in the right foot and saline in the left. After 2 hr, We animals were sacrificed with ether and both legs wen cut offat the ankle and weighed individually . The increase of weight due to oedema was calculated by subtracting from the right leg We weight of the left leg injected with saline. Results are expressed in mglPg of venom. Estimation of coagulant activity

Coagulant activity was determined using citrated horse plasma as substrate. Scrial dilutions keeping a constant ratio of 2 and starting from a concentration of 1 mg/ml of the venoms wen performed. These solutions (100111) were added to 300111 of the plasma and the clotting time assayed at 37°C . The reciprocal of the venom concentration was plotted against the clotting time in order to obtain a straight Gne. Results an expressed as the reciprocal of venom concentration able to clot the plasma in 1 sec. Fstimation of phospholipase A= activity

The method of Mexa~ri?rn (1965) was used for evaluation of phospholipase activity with slight modifications . Egg-yolk was suspended in Tris-HCl buffer, pH 8.0, to an initial absorbance of 1 .0 at 740 nm . One hundred micrograms of each venom was added to 3 ml of this suspension. Phospholipase activity was assessed as the rate

Toxic activities of Bothropa Venons T~~ 1. Tout ecrtvtr~ of venons

Bothrops vexoes

Haemorrhagic Oedema Proteolytic Phospholipasic Necrotizing (mm=/a+g) (mg/ug) N/mg) N/mg) (~~/ue)

B. alternatus B. atrox B. cotiara B. erythromelas B. Jarmaca B. jararacwse B. moojeni B. neuwiedi B. pradoi

12.9 f 4.3 2.2 t 0.2 8.2 t2.5 5.2 t0.9 15 .4 f 1.9 1.1 f 0.1 1.6 f0.5 3.0 t0.5 3.8 t0.7

91 t l6 66 f 11 80 t 4 105 t 18 111 f20 75 t 9 86 t4 86 t 8 92 t 27

0.14 f0.01 0 .42 t 0.01 0.04 t 0.00 0.33 f0.01 0.31 f0.01 0.15f0.02 0.27 f0.01 0 .14 f0.01 0.25 t 0.02

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392 t86 291 t 31 0 753 f45 763t43 517 t 122 843 t 117 805 t 135 819 t 98

Myotoxic (U/m8)

Coagulant (mB')

0.86f0.39 6.9 t 2.6 215 t 42 2.63 f0.47 33 .7 t 13 .1 2125 f 160 0.26f0.25 16.0 t 8.0 249 f 19 3.63 t 1.55 9. I t 5.1 12,744 t 1987 3.30f0.42 10 .0 f 2.5 469 t 23 2.57 f0.49 115.0 f 30 .0 822 f43 1.13 f0.75 103.1 t 19 .5 714 f65 8.37 f 1.70 82 .6 f 35 .6 3380 t 150 7.18 t 1.06 75 .0 t 20 .1 16,167 t 1336

Venom activities were assayed as described in Material and Methods and are expressed as the mean of thra independent experiments tS.D . of a linear decrease in O.D. over an incubation peeriod of 5 to 15 mina . One unit of phospholipase activity corresponds to a decrease of 0.001 of absorbante per min. Activity is expressed as U/mg of venom. 1=rtânatton ojmyotoxic activity

Mice were injected i.m. (thigh muscle) with 200 ug of the venoma of B. alternates, B. cotiara, B. erythromrlas and B. jararaca or 50 pg of the venoms of B. atrox, B. jararacvsae, B. moojeni, B. neewiedl and B. pradoi. After 3 hr, blood was collected from the opthalmic plexus under light ether anaesthesia. Sera from three mice were separated, pooled and immediately assayed for creatin kinase activity (Nao-CK assay system from Merck do Hrasil, S8o Paulo, Hrazil), as described by the manufacturers (~+x Socn rv of Ct.nvic~. G~tszs, 1977). One unit corresponds to the amount of enzyme that hydrolyses 1 tanole of creative per min at 25°C . Myotoxic activity is expressed as U/mg of venom. F .atimatton ojproteolytic activity

Proteolytic activity was estimated using casein as substrate, as described by MANDELBAUM et al. (1982). One millilitre of l'/° casein was incubated for 2hr at 37°C with 400pl of test solutions containing 50 pg of each venom, in the presence of 0.008 M CaClb at pH 8.8. Reaction was stopped with 5°/. TCA and We hydrolysed peptides contained in the supernatants quantified according to LOWRY et al. (1951). One unit is defined as the amount of enzyme yielding an increase in O.D. of 1 .0 per min at 750 nm . Results are expressed in U/mg of venom. Statistical analyses

Correlatos were tested by means of linear regression analysis with thecorrelation coe~cient of Pearson using the Sigmaplot software (Sigma, U.S .A .) and the significance according to Dexm. (1987) . RESULTS

Table 1 shows the distribution of each toxic activity among the nine venoms tested . The haemorrhagic activity was predominant in B. jararaca and B. alternates venoms, while B. jararacesse and B. moojeni venoms showed the lowest levels of this activity . The proteolytic activity was concentrated in B. atrox, B, erythromelas, B. jararaca, B. moojeni and B. pradoi venons. The phospholipase activity was very low in B. alternates and B. atrox venoms and was not detected in B. cotiara venom, even after long periods of incubation of venom and substrate. The necrotizing activity was predominant in B. neuwiedi and B. pradoi venoms, while the myotoxic activity was higher in B. jararacessu, B. moojeni, B. neuwiedi and B. pradoi venous . The coagulant activity was prevalent

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M. L. FERREIRA et al. T~nr p 2 . Coxteat .e~noN ct~tc~rts (R) oP tBrttAt.rtY Axn

rmcreoss wtrti ort~t ectmr~s a~ Bothrops varaxs

Activities Lethality Necrotizing Haemorrhagic Oedema Proteolytic Phospholipasic Myotoxic Coagulant

Lethality

Necxosis

0.34 0.54" 0.60" -0.59" 0.13 -0.21 0.09

0 .34 -0 .28 0 .18 0 .12 0 .62 " 0 .32 0 .58 "

Significant cornlation (P < 0.1) .

in the venoms of B. pradoi and B. erythrotnelas, followed by B. tieuwiedi and B. atrox venoms. All venoms presented high levels of oedema-inducing activity. The values of the toxic activities of each venom were plotted against the values of the LD P or the necrotizing activity of the respective venom. The linear regression of these plots allowed the estimation of the correlation coefficient of the activities in relation to the venom lethality or their ability to induce local necrosis. As shown in Table 2 there was a significant correlation (P < 0.1) between lethality and haemorrhagic or oedema-inducing activities as well as between necrotizing activity and phospholipase or coagulant activities . On the other hand, there was an inverse correlation between the proteolytic activity and lethality. However, none of the activities presented a straight correlation (R > 0.9) with either lethality or necrotizing activity . This may be explained by assuming that the lethality and necrosis of the Bothrops venoms result from the combined actions of the different toxins . DISCUSSION

This study shows that the main toxins present in venoms of Bothrops snakes are not equally distributed among the different species. This observation is similar to that reported for pro-coagulant (NAHAS et al., 1979), necrotizing (VILLARROBL et al., 1978/79), platelet-aggregating and phospholipase (Z1xcALt et al., 1988) activities . However, in our experiments we found values for the coagulant and necrotizing activities of some venoms that differ significantly from the values reported by these authors. These differences are probably due to the methodology employed by the different authors such as the source of plasma to assay the pro-coagulant activity, the experimental model used to evaluate the venom induced necrosis or even to differences in the pools of venoms used. The statistical analyses of the different venom activities showed that the haemorrhage and oedema induced by the venom presented a significant correlation with lethality, whereas the phospholipase and coagulant activities were correlated with necrosis. The observation that there was no straight correlation between any of the toxic activities and necrosis or lethality suggests that the different toxins probably have an additive or synergic effect . It is important to point out that each activity shown here was assayed in the crude venom. Additive or synergic effects of the different toxins may be also occurring in the cases of the oedema-inducing, haemorrhagic and myotoxic activities .

Toxic activities of Bothrops Veaoms

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The clinical implications of these observations must be considered . Haemorrhagic factors present in the venoms are closely related to haemorrhages affecting essential organs, renal insufficiency and shock, that are the most typical symptoms of the severe envenomings followed by death of the patients . On the other hand, oedema does not seem to be directly related to lethality. However, the same components that induce oedema may also be altering some of the essential functions of the patient, thus contributing to the lethality of the venoms . This hypothesis is strongly corroborated by the clinical observations that severe Bothrops accidents always present intense oedema (AMARAL et al., 1991). The correlation between the levels of phospholipase and necrotizing activities is well explained by the role of endogenous phospholipase AZ in the liberation of pro-infiamatory mediators responsible for infiltration and activation of the phagocytic cells (PRUZANSKI and VADAS, 1991). Venom phospholipase A2 may be acting in the same way, increasing the inflammatory reaction in the site of the venom injection and facilitating venom-induced necrosis. The correlation between coagulant activity and necrosis may be explained by the formation of mural thrombi that participate in the settlement of the ischaemia (G~RF~ and LOMONTE, 1989). However, the haemorrhagec and oedema-inducing factors also corroborate for the development of the ischaemia and yet did not show significant correlation with the necrotizing activity. We were unable to find references to the correlation between the coagulant and necrotizing activities in patients. Proteolytic activity, assayed using casein as substrate, showed a significant inverse correlation (P < 0.1) with lethality. However, the meaning of this inverse correlation is poorly understood in the case of venoms of Bothrops species. One possible explanation may be a phenomenon similar to that occurring in the venom of different specimens of Crotalus scutulatus st:uttrlatus. In this case, venoms that have high proteolytic activity contain little amounts of the lethal Mojave toùn and consequently present decreased lethality (GLEt?N and STRAIGHT, 1989). In the case of Bothrops species, some studies on the ontogenetic variability of their venoms showed that proteolytic enzymes present an inverse correlation with some procoagulant factors (FURTADO et al., 1991). However, as shown here the coagulant activity was not related to lethality. It is reasonable to expect that some lethal components of Bothrops venoms are inversely correlated with the proteolytic enzymes, although this is not clear at the moment. The data showing that toxins are differently distributed in the venoms of the different species of Bothrops and that the different toùns are contributing to lethality and necrosis are particularly important regarding antivenom therapy. Venoms from different species probably induce systemic and local effects of different intensities, requiring eventually different clinical procedures . This hypothesis is under investigation at experimental level in our laboratory, by the inspection of mice envenoming by different Bothrops venoms as well as the effectiveness of serum therapy or alternative treatments for those different envenomings. dcknowledgements-This work was supported by FAPESP (process 90/1879-2). M .L.F . was the recipient of a FAPESP fellowship (process 90/2465-7). REFERENCES A~i., C . F . S., Douruno, H . V ., Kouvotnmlux, J . A., C~enaso, J . L ., Ce~os, J. A ., Azßv®o-MNtQups, M . M . and Lor~, P. F. A . (1991) Mmaml de diggnQrtfco e trretmnento de acidentes oJfdicar. Ministério da Safide, Hraailia.

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Toxic activities of venoms from nine Bothrops species and their correlation with lethality and necrosis.

The comparison of seven toxic activities contained in venoms from nine different species of Bothrops and the correlation of each activity with lethali...
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