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Hyperalgesia induced by Bothrops jararaca venom in rats: Role of eicosanoids and platelet activating factor (PAF)
Tackron, Vol. 32, No. 4, pp . 4I9-426, 1944 ~PYdibr © 1944 Ehevier Scknca Ltd Printed in Grat Hrium. All ti~u tnmved 0041-0101/94 56.00+0.00
0041-0101(93)IE002~L
HYPERALGESIA INDUCED BY BOTHROPS JARARACA VENOM IN RATS: ROLE OF EICOSANOIDS AND PLATELET ACTIVATING FACTOR (PAF) C. F. P. TI~mA,' Y. CuRY,s S. OcA' and S. JANCAR4
Laborat6rio de 'Farmacologia e 'Fieiopatologia, Inetituto Butantan, 05503-900, Säo Paulo, SP, Brazil; and Departamento de 3Farmacologia e'Imunolo~s, Instituto de CSências Biomédicas, Universidade de Silo Paulo, 05508-900, Silo Paulo, 3P, Brazil (Recefaed
15 ltou 1993 ;
accepted
9 Nooemlxr 1993)
C. F. P. Ti?ixEmw, Y. CURY, S. OcA and S. JANCAR . Hyperalgesia induced by Bothrops jararaca venom in rats: role of eicosanoids and platelet activating factor (PAF) . Toxicon 32, 419-426, 1994.-In this study we investigated the ability of Bothrops jararaca venom (BjV) to induce hyperalgesia and the modulation of this effect by lipid mediators . It was found that intraplantar injection of BjV (1 to 25 hg) caused a dose and time-related hyperalgesia . The peak of the hyperalgesic response was 1 hr after injection of the venom and persisted for 24 hr with the higher dose. The BjV-induced hyperalgesia was markedly attenuated by dexamethasone . Dexamethasone blocks the generation of biologically active metabolites from arachidonic acid by inhibiting PLA N activation. Inhibition of the cyclo-oxygenase pathway by indomethacin, or inhibition oflipoxygenases by NDGA both significantly inhibited BjV-induced hyperalgesia . Two antagonists of PAF, WEB2170 and BN52021, also significantly inhibited the initial phase of the hyperalgesia . These results suggest that hyperalgesia induced by BjV is, at least partially, mediated by lipid mediators such as prostaglandins, leukotrienes and PAF .
INTRODUCTION
BOTFIROPIC VeIIOIriS (family Viperidae) cause pronounced local effects in man and animals, characterized by hemorrhage, edema, intense local pain and nocrosis (ROSENFELD, 1971 ; Jn~lvsz-PoRItAZ, 1973; GuTl~xEZ et aL, 1981). Such effects are related to the wmbined action of proteases (VTTAL BRAZII., 1982; MANDF.LHAUM et al., 1982), hemorrhagic factors (HoussAY,1930 ; ÀSSAKURA et a1.,19815) and phospholipases (VIRAL et a1., 1972; GuTmzREZ et al., 1984) present in these venoms, as well as to the release of pharmacologically active compounds generated by the venom . Venom phospholipases can trigger important cellular events, among which are the release of arachidonic acid and lysophospholipids from cell membranes. Free arachidonate can then be metabolized by two enzymatic systems, 4;yclo-oxygenase and 5-lipoxygenase, leading respectively to the formation of eicosanoids (prostaglandins, thromboxanes and leukotrienes), and lysophospholipids which can be further transformed into a biologically active compound known as platelet activating factor (PAF). In addition, tissue damage or injury by other venom components can 419
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C . F. P. TEIXEIRA et al .
activate endogenous phospholipases which also result in the release of eicosanoids and PAF. These substances are potent mediators of inflammation and pain, and appear to play a role in the local effects consequent to Bothrops jararaca bite . TRF.BIEN and CALIXTb (1989) have shown that eicosanoids and PAF contribute to the edema induced by injection of Bothrops jararaca venom (BjV) into rat foot pad. Besides edema, inoculation of Bothrops venom into men and animals elicits intense pain (RosErir-eLn, 1971). However, despite its clinical relevance, the study of Bothrops venom-induced pain and hyperalgesia has received little attention, stressing the need for a comprehensive approach to the study of Bothrops venoms-induced pain . The present study was undertaken in order to investigate the hyperalgesic response in rats caused by BjV injection and the contribution of lipid mediators to this venom action . MATERIALS AND METHODS Venom Lyophilized crude venom of Bothrops jararaca (Bj~ was supplied by the Laboratbrio de Herpetologia, Instituto Batsman, Brazil . Evaluation of hyperalgesia Adult male Wistar rats (150-180 g) received either 0 .1 ml of sterile 0.9% NaCI solution (control animals) or 0 .1 ml of this solution containing the appropriate wnantrations of BjV into the subplantar surface of one hind paw . The contralateral paw was not injected . The pain threshold was measured at different times after venom or saline injection using an Ugo-liasile pressure apparatus, essentially as described by Rexnec.c and Ser.rrro (1957) . Briefly, a force with increasing magnitude was applied to the paw. When the animals reacted by withdrawing the paw, the force (in g) needed to induce such response represented the pain threshold. Evaluation of edema Adult male Wistar rats (150-180 g) were injected into the subplantar surface of one hind paw with 0, I ml of 0 .9% NaCI sterile solution containing different concentrations of BjV. The contralateral paw received the same volume of 0,9% NaCI sterile solution . The volume increase (edema) of paws was measured plethysmographically according to the method of Vex Axat~N et al. (1965), at several intervals following Bw injection . The results wcre calculated as the difference between the values obtained in both paws and expressed as % increase in paw volume. Drug treatments To investigate the involvement of arachidonate metabolites on the BjV-induced hyperlagesia different groups of rats were tt~eated with the cyclo-oxygenase inhibitor indomethacin (4 mg/kg, i,v .) or the lipoxygenase inhibitor, nor-dihydroguaiaretic acid (30 mg/kg, i.v .), 30 min before intraplantar injection of 5 pg BjV/paw. Another group of animals was treated with dexamethasone (0.4 mg/kg, p.o .) 90 min before the venom injection in order to evaluate the contribution of endogenous phoapholipase A z activity to the hyperalgesic effect of the venom. Similarly, the contribution of PAF to BjV-induced hyperalgesia was studied by treating groups of refs with the PAF receptor antagonists BN52021 (5 mg/kg, i .v .) and WEB2170 (5 mg/kg, i .v .) 30 min before injection of the venom . The dose of 5 Rg BjV/paw was chosen because it produces a submaximal but reproducible hyperalgesia, enabling the detection of potential inhibition or exacerbation by drug pretreatments . Dn4gs used Indomethacin and nor-dihydrogusiaretic acid (NDGA), were purchased from Sigma Chem. Co . {U .S.A .). Dexamethasone was purchased from Merck (Brazil). BN52021 (3-t-butyl-hexahydro-4,7b,11-trihydroxy-8methyl-9H-1,7a-epoxy-methano-lH,6aH~yclopenta[c]furo(2,3-b] furo-[3',2' : 3,4] cyclopenta [1,2-d] furan-5,9, 12 (4H)trione) and WEB2170(4{(6{o~hlorophenyl}-8,9-dihydro-l-methyl-4H,7 Hcyclopental[4,5]thicno-[3,2-1]-striazolo[4,3-a][1,4]diazcpin-8-yl]carbonyl}morpholine) were kindly supplied by Institut Henri Heaufour (Franc) and Bachringer Ingelhcim (Germany), respectively . Statistical analysis Statistical evaluation of data was carried out by analysis of variance and sequential differences among means according to Tukey contrast analysis at P < 0.05 (Sotut, and RoFn-e, 1981) .
Bothrops jararaca Venom and Hyperalgesia
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RESULTS
Characterization of the hyperalgesia induced by Bothrops jararaca venom The intraplantar injection of BjV (1 .0, 5.0 and 25.0 pg/paw) into the rat hind-paw caused a significant increase in sensitivity to pain (Fig. 1). The intensity of the hyperalgesia induced by 25 Fig BjV was significantly higher than that induced by 5 pg. The effect induced by 5 pg was not statistically different from that produced by 1 pg BjV/paw . The peak of the hyperalgesic response occurred 1 hr after venom injection and remained significantly different from control throughout the time of observation (24 hr). In order to investigate whether BjV-induced hyperalgesia correlated with the edema, both events were compared at the same time intervals . The intraplantar injection of BjV (1 and 5 hg/paw) in the rat caused a dose and time-related edema . The maximum increase in hind-paw swelling occurred at about 1 hr after venom injection. After this time the swelling decreased gradually over the following 5 hr and completely disappeared within 24 hr (Fig. 2). The initial phase (1 hr) of both edematogenic and hyperalgesic responses was similar but the hyperalgesia persisted much longer . Doses of 25 BjV/paw or more caused marked local hemorrhage; in all doses studied the venom did not cause death, at least within the 24 hr observation period (data not shown). E,(%ct of inhibitors of eicosanoidsynthesis on Bothrops jararaca venom-induced hyperalgesia The phospholipase AZ inhibitor, dexamethasone, significantly reduced the rat hind-paw hyperalgesia induced by BjV at the time intervals represented in Fig . 3. The cyclo-oxygenase inhibitor, indomethacin, was also very potent in reducing the hyperalgesia (Fig. 3). The lipoxygenases inhibitor, NDGA, significantly reduced the hyperalgesia at 30 and 120 ~ 100 ~
Time (h) FIa . 1 . EI~ECr of B. faIOlaCa VENOM ON PAIN THRE4FIOLD 1N THE RAT HIND PAW . Threshold response to pressure was measured at different times in the rat lend paw injected with ( " ) saline (control group), crude BjV: (/) l Kg; (~) 5 pg or (~) 25 Rg. Segsitivity to pain was measured as the threshold response to pressure and expressed as g. Each point rept+esents the mean f S.E .M . in a group of 6-10 animals. 'Values are significantly different from control group (P < 0.0~ .
TIIrN (h) Flc. 2 .
COMPARL90N oP Tt>E mst~A-Ixouclxc AND HrPeRACeeslc ePPecls oP RAT HIND PAWS.
B. jararaca
vexo~l
nv
Increase in paw volume (A) and decrease in threshold response (B) were determined at different times in rat hind paws injected with (~) 1 or ( " ) S ug/paw of crude BjV . Data are expressed as percent change in both responses as compared to control paws. Each point is the mean t S .E.M . of 6 animals. +SigniScaatly different from control group (P < 0.05) .
60 min after BjV injection (Fig. 3). However, at longer time periods after BjV injection NDGA was no longer effective (Fig. 3). Bothrops jararaca venom-induced hyperalgesia BjV-induced hyperalgesia was significantly reduced by pretreating the animals with 5 mg/kg of the PAF antagonists BN52021 and WEB2170 (Fig. 4). Both drugs were effective only in the initial phase of venom-induced hyperalgesia . Analgesia induced by WEB2170 was observed in the first 30 min, whereas that induced by BN52021 lasted for 1 hr (Fig. 4). Role of PAF on
DISCUSSION
We have shown that the intraplantar injection of BjV causes a long lasting hyperalgesia. While the hyperalgesia was maximal between 30 and 60 min, it persisted for more than 24 hr . In contrast, BjV-induced edema also reached s maximum 1 hr after injection but was partially reduced at the 5th hr and disappeared completely within 24 hr. Therefore, the time courses of the development and withdrawal of hyperalgesia and edema are dissociated, at least after the 1st hr. In addition, both responses differed markedly with respect to the BjV dose: the hyperalgesic response was similar with doses 1 and 5 ug of BjV, whereas the edematogenic response to the 5 pg dose was significantly higher when compared to the 1 ~g dose. Therefore, it seems that the pain caused by BjV is not a direct consequence of the edema. Release of specific hyperalgesic mediators may be postulated . Participation of inflammatory lipid mediators (prostaglandins, leukotrienes and PAF) in edema induced by BjV has already been described (TYeES>Err and C~Lix~ro, 1989). Hyperalgesia induced by BjV appears to be mediated by eicosanoids since it was markedly reduced by dexamethasone. A possible mechanism of action of this drug seems to be the stimulation of lipocortins synthesis which in turn inhibits phospholipase AZ (Fr.owett and
Bothrops Jararaca Venom and Hyperalgesia 120 ~ 100 -
.~ 40 n. 20 ~
Tlme (h)
Fta. 3. EFFECT OF INIiIHrrORS OF ARACHIDONIC AC07 MEfABOLLL~tt ON TiO: HYPFRAWE4fA INDUCED BY B. ,%ürOI000 VENOAl. ( ") Dexamethasone (0 .4 mg/kg, p.o .) was given 90 min before and (~) indomethacin (0.4 mg/kg, i.v.), (") NDGA (30 mg/kg, i.v .) or (") saline (control group) were given 30 min before injection of S pg of crude BjV/paw. Hyperalgesia was measured as the threshold response to pressure and expressed in g. Each point is the mean f S.E .M . of 6-10 animals. 'Significantly different from control group (P < 0.0~.
120~ 10080 _ ß0 _
20 0
Tlme (h)
Fta . 4. EFFECT of PAF ANi'AOONL415 ON HYPBRALOHSA INDUCED BY B. jararaca vENOM IN nAr tflND PAWa.
The PAF antagonists (") BN52021 and (~) WEH2170 were given i .v. 5 mg/kg, 30 min before injection of 5 pg of B~~/paw. The control group (") received saline i .v . Hyperalgesia was measured as the thn~hold response to pressure and expressed in g. Each point is the mean t S.E .M . of X10 animals. 'Significantly diffet+ent from control group. (P < 0.05) .
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BLACKWELL, 1979). This mechanism prevents generation of the free arachidonic acid needed for the synthesis of eicosanoids. Participation of prostaglandins in the hyperalgesia induced by BjV was further confirmed by the results of the pretreatment of rats with a cyclo-oxygenase inhibitor indomethacin, which markedly reduced the venom-induced pain, Prostaglandins of E series (PGE Z, PGE,) and prostaglandin I2 (PGIZ) are well~stablished hyperalgesic mediators (Ft~mn et al., 1973, 1978). Their ability to sensitize pain receptors to chemical and mechanical stimulation has been demonstrated in humans and animals by different techniques (FERREIRA, 1983). Moreover, our results indicate a contribution of leukotrienes to the initial phase of hyperalgesia induced by BjV since it was significantly reduced by the lipoxygenases inhibitor, NDGA. There is evidence that some 5-lipoxygenase products are important mediators of experimentally induced hyperalgesia, Rncxxna~t and Foxn-Hu~rcfnxsox (1983) and LEVixa et al. (1984) showed that leukotriene B, causes hyperalgesia when injected into rat hind paws . Our results also implicate PAF as a mediator of BjV-induced hyperalgesia since it was significantly reduced by two specific antagonists of PAF, the ginkgolide BN52021 and the hetrazepinoid WEB2170 . It is known that PAF injected subcutaneously into rat paws, causes edema and hyperalgesia of rapid onset and short duration (VnxcwFnc and Ft xx~n, 1981 ; O'Fr.~~rx et a1.,1982 ; BI~Quer et al., 1987). This hyperalgesic response is dependent on the synthesis of leukotrienes (Dnr i.os et al., 1987). The mechanisms by which BjV induces release of eicosanoids and PAF are not known at this time . One possibility is that components of the BjV, such as PLAZ (MOUxw nA SILVA et al., 1991) acting on membrane phospholipids, will release arachidonic acid and the corresponding lysophospholipid which would then be transformed into edematogenic and hyperalgesic substances such as prostaglandins, leukotrienes and PAF. In fact, HAWOR~rx et al. (1988) demonstrated that injection of Naja naja PLAZ in rat paw caused hyperalgesia and edema which were mediated by cyclo-oxygenase and lipoxygenase metabolites of arachidonic acid, respectively . Thus, venom PLAZ may have an important pathogenic role in the development of local effects induced by venoms . Furthermore, other components of the venom can activate endogenous phospholipases with subsequent release of arachidonic acid and lysophospholipids and formation of biologically active metabolites. The strong inhibition of the BjV-induced hyperalgesia by dexamethasone reported in this study is consistent with this latter hypothesis. Bothrops jararaca venom also contains proteases and hemorrhagic toxins which may contribute to the development of hyperalgesia . Venom proteases acting on plasma kininogens release bradykinin (Rocxn E Sn.v~ et al., 1949 ; RoTxsct~.u and RO~scFULn, 1979). Moreover, hemorrhagic toxins by disrupting the microvasculature may induce extravasation of plasma (OHSnxn, 1979) and activation of the coagulation cascade leading to the formation of kinins . It is well established that bradykinin is an important mediator of hyperalgesia and pain . COLLIER et al. (1968) showed that intraperitoneal injection of bradykinin induces pain which was inhibited by non-steroidal anti-inflammatory drugs. This indicates that the pain-inducing effect of bradykinin is mediated by cyclo-oxygenase metabolites. Infusion of bradykinin into guinea-pig lung induces the release of prostaglandins and related substances (PIPER and VnxE, 1969). Several experimental lines of evidence have clearly established that bradykinin causes release of prostanoids and that some effects of bradykinin are mediated by these metabolites (TERRnaxo and TERRncxo, 1979). Therefore, our results with indomethacin suggest that bradykinin, by releasing prostaglandins, contributes to the hyperalgesic effect of BjV. Further studies are required to
Bothrops,jararaca Venom and Hyperalgesia
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investigate this hypothesis. In addition, bradykinin has been shown to release algogenic mediators, such histamine, from mast cells (Joxxsox and ERnôs, 1973 ; ISHIZAKA et al., 1985), thereby enhancing its algesic effect. It is known that BjV induces release ofhistamine (ROTFISCaüLI) and ROTHSCHILD, 1979) . Therefore, the role of histamine on venom-induced hyperalgesia deserves further investigation. The present study provided the first experimental evidence that BjV causes hyperalgesia and that prostaglandins, leukotrienes and PAF contribute to this effect . The generation of a wide array of pharmacologically active compounds that can be induced by BjV and the possible synergic interactions between them should be considered when studying venom-induced effects. Ackrwwkdgiements-The authors thank Professor Zut~ P . PtcnxELU (Instituto Batsman) for her comments on the manuscript, Ix~ PEnxaso VtEttee (LJSP) for valuable technical assistance and Cttuctc Sttn~te Fete~tt for language revision . REFERENCES AssAKUx~, M . T., RQC~u., A . P. and MexnELaeu~t, F. R. (1986) Comparison of immunological, biochemical and
biophysical properties of the three hemorrhagic factors isolated from the venom of Bothrops jararaca (jararaca). Toxicon 24, 943-946 . BxeQuEr, P., TouQut, L ., St~tv, T. Y. and VNea~nc, B . B . (1987) Perspoctives in platelet activating factor. Pharntac . Rev. 39, 97-145 . CoLLtste, H . O . J ., Dtxxtw, L . C., Joxtvsox, C . A . and ScFnvanstt, C. (1968) The abdominal constriction response and its suppression by analgesic drugs in the mouse . Br . J. Pharmac . 32, 295-310 . DeLt,os, A., Gutxnox, Y . and GoLnExaEac, M . M . (1987) Pharmacological evidence for a role of lipoxygenase products in platelet-activating factor (PAF induced hyperalgesia. Biochem. Pharmac. 36, 3201-3204. F~tex, S . H. (1983) Prostaglandins, peripheral and central analgesia . In : ~ldvmtces in Pain Research and Therapy, Vol . V, pp . 62734 (Boxten, J . J ., Ed .). New York: Raven Press . FY~QQ~ " , S. H ., Moxcnne, S. and V~xE, J. R . (1973) Prostaglandins and the mechanism of analgesia produced by aspirin-like drugs. Br . J. Pharmac. 49, 8697. F~aaEtne, S . H ., Nex~ux~, M. and Cestno, M . S. A. (1978) The hyperalgesic effects of proatacyclin and prostaglandin E_ . Prostaglandins 16, 31-37. FLOwEtt, R. J . and HLectcwELL, G. J . (1979) Anti-inflammatory steroids induce biosynthesis of a phospholipase Az inhibitor which prevents prostaglandin generation . Nature, London 278, 45659 . G1iTIFR4F7, J . M., GtAVES, F., Hot .Axos, R ., CEanns, L., Rwns, E ., AweoYO, O. and PORTTLLA, E. (1981) Neutralization de los efectos locales del veneno de Bothrops riper por un antiveneno polivalente. Toxicon 19, 493-500 . GuTmttteEZ, J . M ., Owxsv, C . L. and ODELL, G. V . (1984) Isolation of a myotoxin from B. riper venom: partial characterization and action on skeletal muscle . Toxicon 22, 115-128 . HAWORTH, D ., HEaox, J . R . M . and CexEtr, F . (1988) Rat paw hyperalgesia and oedema in response to NMDA Naja naja phospholipase A a . Br . J. Pharmac . 93, 145 . HOttssw, B. A. (1930) Classification des actions des venins de serpents sur (organisme animal . C. R. Soc . Bial. Paris 105, 308-310 . ISHIZAKA, T ., Iw~rw, M . and L9tttzetu, K . (1985) Release of histamine and atachidonate from mouse mast cells induced by glycosylation-enhancing factor and bradykinin. J. Immunol. 134, 1880-1887 . Jn~xez-Poate~s, J. M . (1973) Reptile toxins. In : Biology Data Book, 2nd Edn, Vol. II, p . 697 . Federation of American Societies for Experimental Biology (FASEH), Bethesda, MD, U.S .A . Jottxsox, A . R. and EtinBs, E. G . (1973) Release of histamine from mast cells by vasoactive peptides. Proc. Soc. exp. Biol. Med. 142, 1252-1256. LEVtxE, J. D ., Lew, W., Kw~tr, G . and GatazL, E. J . (1984) Leukotriene B, produces hyperalgesia that is dependent on polymorphonuclear leukocytes. Science 225, 743-745 . MANDELBAUM, F . R ., RIICttL, A. P. and Ass~teute~, M . T . (1982) Isolation and characterization of a proteolytic enzyme from the vcaom of a snake Bothrops jararaca (jararaca). Toxicon 20, 955-972. Moutee-D~-Stt.ve, A. M., DESMOND, H ., L~txo, G . and Tl~tcsrox, R . D . G. (1991) Isolation and comparison of myotoxins isolated from venoms of difl'erent species of Bothrops snakes. Toxicon 29, 713-726. O'Fut~x~rtr, J . T., WYICLE, R . L ., Txontes, M . J . and MCCALL, C . E . (1984) Neutrophil degraaulation responses to combinations of arachidonate metabolites and platelet-activating factor. Res. Comm+m. Chem . Path. Pharmac. 43, 3-23 .
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OxsAlu, A. (1979) Hemorrhagic, necrotizing and edema-forming effects of snake venons. In : Snake Venons, pp . 480-546 (Lrai, C . Y ., Ed .) . Berlin: Springer. PtrEa, P. J . and VANe, J . R . (1969) Additional factors in anaphylaxis and its antagonism by antünflammatory drugs . Natwe 273, 29-35 . RAC~IAir, A . and Foao-HurcEUxsox, A . W. (1983) Inflammation and pain sensitivity : effects of leukotrienea D B, and prostaglandin E, in the rat paw . Prostaglandins 25, 193-203. RANnAI.r., L. O . and Sel.rno, J . J . (1957) A method for measurement of analgesic activity on inlianed tissue. Arch . Int . Pharmacodyn . 111, 209-219 . ROCHA E Su .vA, M ., HeleAl.no, W . T . and Ro~Nl+al~, G. (1949) Bradykinin a hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venom and by trypain . .lm. !. Physiol. 156, 261-273 . Ro~rn, G. (1971) Symptomatology, pathology and treatment of snake bites in South America . In: Veaommrs Minsk and 77uir Venons, Vol . II, pp. 345-362 (BUCI~II'RL, W . and Bvctcl.etr, E . E., Eds) . New York: Academic Press. ROTIISCInI,O, A. M . and ItoTl~lsClßl.n, Z. (1979) Liberation of pharmacologically active substances by snake venons . In: Handbook of Experimental Pharmacology, Vol. 52, pp . 59128 (L~, C. Y., Ed.) . New York: Springer . Soe"~, R. R . and RoIn.F, F. J . (1981) Biometry, 2nd Edn, 859 pp. New York: W. H . Freeman and Co . TcaaAONO, N. A . and TExaAONO, A. (1979) Release of vasoactive substances by kinina. In: BradykBlhl Kallidin and Kallikrein, pp. 40126 (Fatn6s, E. G., Ed .) . New York: Springer. TxESmN, H. and CAUxro, J . H . (1989) Pharmacological evaluation of rat paw oedema induced by Bothrops jararaca venom . Agents Actions 26, 292-300. VAN ARe~AN, C . G., BeOArnr, A. J ., Mn.l .aa, L . M. and PaESS, H. H . (1965) Some details of inflammations caused by yeast and carrageenan . J. Pharnac . exp . Then . 150, 328-334. VAaOAIrnO, B. B. and F7~rta>mtA, S. H. (l9ß1) Blockade of the inflammatory effects of platelet-activating factor by cyclo-oxygenaee inhibitors . Braz . J. Med. Biol. Res. 14, 187-189. VInAI ., J. C ., MouNA, H. 8IId STOPPANT, A . O . M . (1972) A general procedure for the isolation and purification of phoapholipase A z isoenzymes from Bothrops venom. Acts Physiol . Latino Am. 23, 91-109 . VrrAi-B1tAZlr, O. (1982) Peçonhas. In : Farmacodindnica, 6th Edn, p. 1044 (Coae~l-r, C. E ., Ed .) . Rio de Janeiro : Guanabara Koogan .
0041-0101(93)IE002~L
HYPERALGESIA INDUCED BY BOTHROPS JARARACA VENOM IN RATS: ROLE OF EICOSANOIDS AND PLATELET ACTIVATING FACTOR (PAF) C. F. P. TI~mA,' Y. CuRY,s S. OcA' and S. JANCAR4
Laborat6rio de 'Farmacologia e 'Fieiopatologia, Inetituto Butantan, 05503-900, Säo Paulo, SP, Brazil; and Departamento de 3Farmacologia e'Imunolo~s, Instituto de CSências Biomédicas, Universidade de Silo Paulo, 05508-900, Silo Paulo, 3P, Brazil (Recefaed
15 ltou 1993 ;
accepted
9 Nooemlxr 1993)
C. F. P. Ti?ixEmw, Y. CURY, S. OcA and S. JANCAR . Hyperalgesia induced by Bothrops jararaca venom in rats: role of eicosanoids and platelet activating factor (PAF) . Toxicon 32, 419-426, 1994.-In this study we investigated the ability of Bothrops jararaca venom (BjV) to induce hyperalgesia and the modulation of this effect by lipid mediators . It was found that intraplantar injection of BjV (1 to 25 hg) caused a dose and time-related hyperalgesia . The peak of the hyperalgesic response was 1 hr after injection of the venom and persisted for 24 hr with the higher dose. The BjV-induced hyperalgesia was markedly attenuated by dexamethasone . Dexamethasone blocks the generation of biologically active metabolites from arachidonic acid by inhibiting PLA N activation. Inhibition of the cyclo-oxygenase pathway by indomethacin, or inhibition oflipoxygenases by NDGA both significantly inhibited BjV-induced hyperalgesia . Two antagonists of PAF, WEB2170 and BN52021, also significantly inhibited the initial phase of the hyperalgesia . These results suggest that hyperalgesia induced by BjV is, at least partially, mediated by lipid mediators such as prostaglandins, leukotrienes and PAF .
INTRODUCTION
BOTFIROPIC VeIIOIriS (family Viperidae) cause pronounced local effects in man and animals, characterized by hemorrhage, edema, intense local pain and nocrosis (ROSENFELD, 1971 ; Jn~lvsz-PoRItAZ, 1973; GuTl~xEZ et aL, 1981). Such effects are related to the wmbined action of proteases (VTTAL BRAZII., 1982; MANDF.LHAUM et al., 1982), hemorrhagic factors (HoussAY,1930 ; ÀSSAKURA et a1.,19815) and phospholipases (VIRAL et a1., 1972; GuTmzREZ et al., 1984) present in these venoms, as well as to the release of pharmacologically active compounds generated by the venom . Venom phospholipases can trigger important cellular events, among which are the release of arachidonic acid and lysophospholipids from cell membranes. Free arachidonate can then be metabolized by two enzymatic systems, 4;yclo-oxygenase and 5-lipoxygenase, leading respectively to the formation of eicosanoids (prostaglandins, thromboxanes and leukotrienes), and lysophospholipids which can be further transformed into a biologically active compound known as platelet activating factor (PAF). In addition, tissue damage or injury by other venom components can 419
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C . F. P. TEIXEIRA et al .
activate endogenous phospholipases which also result in the release of eicosanoids and PAF. These substances are potent mediators of inflammation and pain, and appear to play a role in the local effects consequent to Bothrops jararaca bite . TRF.BIEN and CALIXTb (1989) have shown that eicosanoids and PAF contribute to the edema induced by injection of Bothrops jararaca venom (BjV) into rat foot pad. Besides edema, inoculation of Bothrops venom into men and animals elicits intense pain (RosErir-eLn, 1971). However, despite its clinical relevance, the study of Bothrops venom-induced pain and hyperalgesia has received little attention, stressing the need for a comprehensive approach to the study of Bothrops venoms-induced pain . The present study was undertaken in order to investigate the hyperalgesic response in rats caused by BjV injection and the contribution of lipid mediators to this venom action . MATERIALS AND METHODS Venom Lyophilized crude venom of Bothrops jararaca (Bj~ was supplied by the Laboratbrio de Herpetologia, Instituto Batsman, Brazil . Evaluation of hyperalgesia Adult male Wistar rats (150-180 g) received either 0 .1 ml of sterile 0.9% NaCI solution (control animals) or 0 .1 ml of this solution containing the appropriate wnantrations of BjV into the subplantar surface of one hind paw . The contralateral paw was not injected . The pain threshold was measured at different times after venom or saline injection using an Ugo-liasile pressure apparatus, essentially as described by Rexnec.c and Ser.rrro (1957) . Briefly, a force with increasing magnitude was applied to the paw. When the animals reacted by withdrawing the paw, the force (in g) needed to induce such response represented the pain threshold. Evaluation of edema Adult male Wistar rats (150-180 g) were injected into the subplantar surface of one hind paw with 0, I ml of 0 .9% NaCI sterile solution containing different concentrations of BjV. The contralateral paw received the same volume of 0,9% NaCI sterile solution . The volume increase (edema) of paws was measured plethysmographically according to the method of Vex Axat~N et al. (1965), at several intervals following Bw injection . The results wcre calculated as the difference between the values obtained in both paws and expressed as % increase in paw volume. Drug treatments To investigate the involvement of arachidonate metabolites on the BjV-induced hyperlagesia different groups of rats were tt~eated with the cyclo-oxygenase inhibitor indomethacin (4 mg/kg, i,v .) or the lipoxygenase inhibitor, nor-dihydroguaiaretic acid (30 mg/kg, i.v .), 30 min before intraplantar injection of 5 pg BjV/paw. Another group of animals was treated with dexamethasone (0.4 mg/kg, p.o .) 90 min before the venom injection in order to evaluate the contribution of endogenous phoapholipase A z activity to the hyperalgesic effect of the venom. Similarly, the contribution of PAF to BjV-induced hyperalgesia was studied by treating groups of refs with the PAF receptor antagonists BN52021 (5 mg/kg, i .v .) and WEB2170 (5 mg/kg, i .v .) 30 min before injection of the venom . The dose of 5 Rg BjV/paw was chosen because it produces a submaximal but reproducible hyperalgesia, enabling the detection of potential inhibition or exacerbation by drug pretreatments . Dn4gs used Indomethacin and nor-dihydrogusiaretic acid (NDGA), were purchased from Sigma Chem. Co . {U .S.A .). Dexamethasone was purchased from Merck (Brazil). BN52021 (3-t-butyl-hexahydro-4,7b,11-trihydroxy-8methyl-9H-1,7a-epoxy-methano-lH,6aH~yclopenta[c]furo(2,3-b] furo-[3',2' : 3,4] cyclopenta [1,2-d] furan-5,9, 12 (4H)trione) and WEB2170(4{(6{o~hlorophenyl}-8,9-dihydro-l-methyl-4H,7 Hcyclopental[4,5]thicno-[3,2-1]-striazolo[4,3-a][1,4]diazcpin-8-yl]carbonyl}morpholine) were kindly supplied by Institut Henri Heaufour (Franc) and Bachringer Ingelhcim (Germany), respectively . Statistical analysis Statistical evaluation of data was carried out by analysis of variance and sequential differences among means according to Tukey contrast analysis at P < 0.05 (Sotut, and RoFn-e, 1981) .
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RESULTS
Characterization of the hyperalgesia induced by Bothrops jararaca venom The intraplantar injection of BjV (1 .0, 5.0 and 25.0 pg/paw) into the rat hind-paw caused a significant increase in sensitivity to pain (Fig. 1). The intensity of the hyperalgesia induced by 25 Fig BjV was significantly higher than that induced by 5 pg. The effect induced by 5 pg was not statistically different from that produced by 1 pg BjV/paw . The peak of the hyperalgesic response occurred 1 hr after venom injection and remained significantly different from control throughout the time of observation (24 hr). In order to investigate whether BjV-induced hyperalgesia correlated with the edema, both events were compared at the same time intervals . The intraplantar injection of BjV (1 and 5 hg/paw) in the rat caused a dose and time-related edema . The maximum increase in hind-paw swelling occurred at about 1 hr after venom injection. After this time the swelling decreased gradually over the following 5 hr and completely disappeared within 24 hr (Fig. 2). The initial phase (1 hr) of both edematogenic and hyperalgesic responses was similar but the hyperalgesia persisted much longer . Doses of 25 BjV/paw or more caused marked local hemorrhage; in all doses studied the venom did not cause death, at least within the 24 hr observation period (data not shown). E,(%ct of inhibitors of eicosanoidsynthesis on Bothrops jararaca venom-induced hyperalgesia The phospholipase AZ inhibitor, dexamethasone, significantly reduced the rat hind-paw hyperalgesia induced by BjV at the time intervals represented in Fig . 3. The cyclo-oxygenase inhibitor, indomethacin, was also very potent in reducing the hyperalgesia (Fig. 3). The lipoxygenases inhibitor, NDGA, significantly reduced the hyperalgesia at 30 and 120 ~ 100 ~
Time (h) FIa . 1 . EI~ECr of B. faIOlaCa VENOM ON PAIN THRE4FIOLD 1N THE RAT HIND PAW . Threshold response to pressure was measured at different times in the rat lend paw injected with ( " ) saline (control group), crude BjV: (/) l Kg; (~) 5 pg or (~) 25 Rg. Segsitivity to pain was measured as the threshold response to pressure and expressed as g. Each point rept+esents the mean f S.E .M . in a group of 6-10 animals. 'Values are significantly different from control group (P < 0.0~ .
TIIrN (h) Flc. 2 .
COMPARL90N oP Tt>E mst~A-Ixouclxc AND HrPeRACeeslc ePPecls oP RAT HIND PAWS.
B. jararaca
vexo~l
nv
Increase in paw volume (A) and decrease in threshold response (B) were determined at different times in rat hind paws injected with (~) 1 or ( " ) S ug/paw of crude BjV . Data are expressed as percent change in both responses as compared to control paws. Each point is the mean t S .E.M . of 6 animals. +SigniScaatly different from control group (P < 0.05) .
60 min after BjV injection (Fig. 3). However, at longer time periods after BjV injection NDGA was no longer effective (Fig. 3). Bothrops jararaca venom-induced hyperalgesia BjV-induced hyperalgesia was significantly reduced by pretreating the animals with 5 mg/kg of the PAF antagonists BN52021 and WEB2170 (Fig. 4). Both drugs were effective only in the initial phase of venom-induced hyperalgesia . Analgesia induced by WEB2170 was observed in the first 30 min, whereas that induced by BN52021 lasted for 1 hr (Fig. 4). Role of PAF on
DISCUSSION
We have shown that the intraplantar injection of BjV causes a long lasting hyperalgesia. While the hyperalgesia was maximal between 30 and 60 min, it persisted for more than 24 hr . In contrast, BjV-induced edema also reached s maximum 1 hr after injection but was partially reduced at the 5th hr and disappeared completely within 24 hr. Therefore, the time courses of the development and withdrawal of hyperalgesia and edema are dissociated, at least after the 1st hr. In addition, both responses differed markedly with respect to the BjV dose: the hyperalgesic response was similar with doses 1 and 5 ug of BjV, whereas the edematogenic response to the 5 pg dose was significantly higher when compared to the 1 ~g dose. Therefore, it seems that the pain caused by BjV is not a direct consequence of the edema. Release of specific hyperalgesic mediators may be postulated . Participation of inflammatory lipid mediators (prostaglandins, leukotrienes and PAF) in edema induced by BjV has already been described (TYeES>Err and C~Lix~ro, 1989). Hyperalgesia induced by BjV appears to be mediated by eicosanoids since it was markedly reduced by dexamethasone. A possible mechanism of action of this drug seems to be the stimulation of lipocortins synthesis which in turn inhibits phospholipase AZ (Fr.owett and
Bothrops Jararaca Venom and Hyperalgesia 120 ~ 100 -
.~ 40 n. 20 ~
Tlme (h)
Fta. 3. EFFECT OF INIiIHrrORS OF ARACHIDONIC AC07 MEfABOLLL~tt ON TiO: HYPFRAWE4fA INDUCED BY B. ,%ürOI000 VENOAl. ( ") Dexamethasone (0 .4 mg/kg, p.o .) was given 90 min before and (~) indomethacin (0.4 mg/kg, i.v.), (") NDGA (30 mg/kg, i.v .) or (") saline (control group) were given 30 min before injection of S pg of crude BjV/paw. Hyperalgesia was measured as the threshold response to pressure and expressed in g. Each point is the mean f S.E .M . of 6-10 animals. 'Significantly different from control group (P < 0.0~.
120~ 10080 _ ß0 _
20 0
Tlme (h)
Fta . 4. EFFECT of PAF ANi'AOONL415 ON HYPBRALOHSA INDUCED BY B. jararaca vENOM IN nAr tflND PAWa.
The PAF antagonists (") BN52021 and (~) WEH2170 were given i .v. 5 mg/kg, 30 min before injection of 5 pg of B~~/paw. The control group (") received saline i .v . Hyperalgesia was measured as the thn~hold response to pressure and expressed in g. Each point is the mean t S.E .M . of X10 animals. 'Significantly diffet+ent from control group. (P < 0.05) .
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BLACKWELL, 1979). This mechanism prevents generation of the free arachidonic acid needed for the synthesis of eicosanoids. Participation of prostaglandins in the hyperalgesia induced by BjV was further confirmed by the results of the pretreatment of rats with a cyclo-oxygenase inhibitor indomethacin, which markedly reduced the venom-induced pain, Prostaglandins of E series (PGE Z, PGE,) and prostaglandin I2 (PGIZ) are well~stablished hyperalgesic mediators (Ft~mn et al., 1973, 1978). Their ability to sensitize pain receptors to chemical and mechanical stimulation has been demonstrated in humans and animals by different techniques (FERREIRA, 1983). Moreover, our results indicate a contribution of leukotrienes to the initial phase of hyperalgesia induced by BjV since it was significantly reduced by the lipoxygenases inhibitor, NDGA. There is evidence that some 5-lipoxygenase products are important mediators of experimentally induced hyperalgesia, Rncxxna~t and Foxn-Hu~rcfnxsox (1983) and LEVixa et al. (1984) showed that leukotriene B, causes hyperalgesia when injected into rat hind paws . Our results also implicate PAF as a mediator of BjV-induced hyperalgesia since it was significantly reduced by two specific antagonists of PAF, the ginkgolide BN52021 and the hetrazepinoid WEB2170 . It is known that PAF injected subcutaneously into rat paws, causes edema and hyperalgesia of rapid onset and short duration (VnxcwFnc and Ft xx~n, 1981 ; O'Fr.~~rx et a1.,1982 ; BI~Quer et al., 1987). This hyperalgesic response is dependent on the synthesis of leukotrienes (Dnr i.os et al., 1987). The mechanisms by which BjV induces release of eicosanoids and PAF are not known at this time . One possibility is that components of the BjV, such as PLAZ (MOUxw nA SILVA et al., 1991) acting on membrane phospholipids, will release arachidonic acid and the corresponding lysophospholipid which would then be transformed into edematogenic and hyperalgesic substances such as prostaglandins, leukotrienes and PAF. In fact, HAWOR~rx et al. (1988) demonstrated that injection of Naja naja PLAZ in rat paw caused hyperalgesia and edema which were mediated by cyclo-oxygenase and lipoxygenase metabolites of arachidonic acid, respectively . Thus, venom PLAZ may have an important pathogenic role in the development of local effects induced by venoms . Furthermore, other components of the venom can activate endogenous phospholipases with subsequent release of arachidonic acid and lysophospholipids and formation of biologically active metabolites. The strong inhibition of the BjV-induced hyperalgesia by dexamethasone reported in this study is consistent with this latter hypothesis. Bothrops jararaca venom also contains proteases and hemorrhagic toxins which may contribute to the development of hyperalgesia . Venom proteases acting on plasma kininogens release bradykinin (Rocxn E Sn.v~ et al., 1949 ; RoTxsct~.u and RO~scFULn, 1979). Moreover, hemorrhagic toxins by disrupting the microvasculature may induce extravasation of plasma (OHSnxn, 1979) and activation of the coagulation cascade leading to the formation of kinins . It is well established that bradykinin is an important mediator of hyperalgesia and pain . COLLIER et al. (1968) showed that intraperitoneal injection of bradykinin induces pain which was inhibited by non-steroidal anti-inflammatory drugs. This indicates that the pain-inducing effect of bradykinin is mediated by cyclo-oxygenase metabolites. Infusion of bradykinin into guinea-pig lung induces the release of prostaglandins and related substances (PIPER and VnxE, 1969). Several experimental lines of evidence have clearly established that bradykinin causes release of prostanoids and that some effects of bradykinin are mediated by these metabolites (TERRnaxo and TERRncxo, 1979). Therefore, our results with indomethacin suggest that bradykinin, by releasing prostaglandins, contributes to the hyperalgesic effect of BjV. Further studies are required to
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investigate this hypothesis. In addition, bradykinin has been shown to release algogenic mediators, such histamine, from mast cells (Joxxsox and ERnôs, 1973 ; ISHIZAKA et al., 1985), thereby enhancing its algesic effect. It is known that BjV induces release ofhistamine (ROTFISCaüLI) and ROTHSCHILD, 1979) . Therefore, the role of histamine on venom-induced hyperalgesia deserves further investigation. The present study provided the first experimental evidence that BjV causes hyperalgesia and that prostaglandins, leukotrienes and PAF contribute to this effect . The generation of a wide array of pharmacologically active compounds that can be induced by BjV and the possible synergic interactions between them should be considered when studying venom-induced effects. Ackrwwkdgiements-The authors thank Professor Zut~ P . PtcnxELU (Instituto Batsman) for her comments on the manuscript, Ix~ PEnxaso VtEttee (LJSP) for valuable technical assistance and Cttuctc Sttn~te Fete~tt for language revision . REFERENCES AssAKUx~, M . T., RQC~u., A . P. and MexnELaeu~t, F. R. (1986) Comparison of immunological, biochemical and
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