ARTICLE IN PRESS

0041-0101/$ - se

doi:10.1016/j.to

Abbreviations

jararaca venom�Correspondi

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Shishima Hifum

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Toxicon 49 (2007) 670–677

www.elsevier.com/locate/toxicon

Role of cyclooxygenases in oedema-forming activity ofbothropic venoms

Renata do A. Olivoa, Catarina F.P. Teixeiraa, John L. Wallaceb,Jose M. Gutierrezc, Stella R. Zamunera,b,�

aLaboratory of Pharmacology, Butantan Institute, Avenida Vital Brazil 1500, 05503-900 Sao Paulo, SP, BrazilbMucosal Inflammation Research Group, University of Calgary, Calgary, Alberta T2N 4N1, CanadacClodomiro Picado Institute, Faculty of Microbiology, University of Costa Rica, San Jose, Costa Rica

Received 2 June 2006; received in revised form 8 November 2006; accepted 13 November 2006

Available online 21 November 2006

Abstract

The venoms of Bothrops asper (BaV) and Bothrops jararaca (BjV), two of the most medically important poisonous

snakes of Latin America, cause pronounced oedema in the victims through poorly understood mechanisms. In the present

study, we examined the possible role of cyclooxygenases (COX) in the genesis of mouse paw oedema caused by BaV and

BjV injections. BaV at 2.5 mg/paw and BjV at 0.75 mg/paw induced significant oedema that persisted for up to 6 h following

subplantar injection. Treatment with indomethacin (2mg/kg), rofecoxib, (10mg/kg), or dexamethasone (2mg/kg)

significantly reduced the BaV- and BjV-induced oedema formation. Treatment with SC-560 (30mg/kg) significantly

reduced the oedema formation induced by BjV but had no effect on that induced by BaV. Both venoms induced significant

increases in the levels of prostaglandin E2 (PGE2) and the expression of COX-1 and COX-2 in paw tissue. The peak of

oedema formation and PGE2 release correlated with marked expression of COX-2 in the paw tissue. These results

demonstrate that injection of BaV and BjV results in a rapid increase in oedema formation that is, at least partially,

mediated by arachidonic acid metabolites formed by COX-2. In the case of BjV, COX-1-derived prostanoids also appear

to contribute significantly to the inflammatory changes.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Bothrops asper; Bothrops jararaca; Cyclooxygenase; Inflammation; COX-2; PGE2

1. Introduction

Snake venoms are rich sources of enzyme-generating factors and enzymes capable of mediat-

e front matter r 2006 Elsevier Ltd. All rights reserved

xicon.2006.11.006

: BaV, Bothrops asper venom; BjV, Bothrops

; COX, cyclooxygenase; PG, prostaglandin

ng author. Universidade do Vale do Paraıba-

uto de Pesquisa e Desenvolvimento—IP&D, Av.

i, 2911 Bairro Urbanova, CEP 12244-000 Sao

el.: +55 1239471106.

ss: szamuner@univap.br (S.R. Zamuner).

ing, directly or indirectly, the early events of thehemorrhagic and inflammatory processes (Rodri-gues et al., 2004). Envenomation by snakes of thegenus Bothrops induces both local and systemicpathological alterations, which are due to theadditive or synergistic effects of the different toxinsand enzymes present in the venoms (Ownby et al.,1982; Gutierrez and Lomonte, 1995; Trebien andCalixto, 1989). Bothrops asper (BaV) and Bothrops

jararaca (BjV) venoms are responsible formost snake poisonings in Costa Rica and Brazil,

.

ARTICLE IN PRESSR.A. Olivo et al. / Toxicon 49 (2007) 670–677 671

respectively. These venoms induce prominent localeffects which include pain, myonecrosis and inflam-mation (Rosenfeld, 1971; Gutierrez et al., 1984;Trebien and Calixto, 1989; Chacur et al., 2001;Zamuner et al., 2001). This inflammatory reaction ischaracterized by oedema formation and leukocyteinflux, which are mediated by histamine, serotonin,complement-derived factors, cyclo- and lipo-oxyge-nase metabolites and cytokines (Trebien andCalixto, 1989; Chaves et al., 1995; Farsky et al.,1997; Zamuner et al., 2001; Zamuner and Teixeira,2002). The oedema caused by these venoms isrelated, at least in part, to their ability to stimulatethe release of eicosanoids (Trebien and Calixto,1989; Chaves et al., 1995).

The eicosanoids prostaglandins (PGs) have arange of biological activities which are importantin physiological and pathophysiological processeswhich include inflammation (Mitchell et al., 1995;Portanova et al., 1996; Smith et al., 1998).Prostaglandin E2 (PGE2) is a major product atinflammatory sites where it contributes to localincreases in blood flow, oedema formation and painsensitization (Giuliano and Warner, 2002). Cy-clooxygenase (COX) is the enzyme that catalyzesthe biosynthesis of the PGs from the substratearachidonic acid (Vane et al., 1998). There are atleast two distinct isoforms of COX (COX-1 andCOX-2) that have differences in structural andkinetic properties (Morita et al., 1995; Murakamiet al., 1999). COX-1 is responsible for the produc-tion of PGs with general housekeeping functionssuch as maintenance of renal perfusion and aprotective effect on the gastric mucosa againstulceration. COX-2 is induced at sites of inflamma-tion but is also constitutively expressed in sometissues, such as kidney and brain (Sakamoto, 1998;Jackson et al., 2000; Porcher et al., 2002).

While there is evidence for involvement of COXproducts in Bothrops spp. venom-induced oedemaformation, the relative importance of COX-1 versusCOX-2 has not been discerned. In this study, wehave examined the contribution of PGs and of thetwo COX isoforms to oedema formation followingadministration of BaV and BjV.

2. Material and methods

2.1. Venom

Lyophilized crude BaV and BjV were supplied byClodomiro Picado Institute, San Jose, Costa Rica,

and the Herpetology Laboratory of the ButantanInstitute, Sao Paulo, Brazil, respectively. Thevenoms were dissolved in 0.15M NaCl solutionand subsequently filtered through sterilizing mem-branes (0.22 mm pore size; Millipore Ind. Com. Ltd.,Brazil) before use.

2.2. Animals

Male, Swiss mice (19–20 g) were used. The micewere housed in temperature-controlled rooms andreceived water and food ad libitum until used. Thesestudies were approved by the Experimental AnimalsCommittee of Butantan Institute (protocol no. 014/2001) in accordance with the procedures approvedby the Universities Federation for Animal Welfare.

2.3. Evaluation of oedema

Fifty microliters of sterile 0.9% (w/v) salinecontaining 2.5 mg of BaV or 0.75 mg of BjV wereinjected in the subplantar region of the right hindpaw. The left hind paw received an equal volume ofsterile saline alone and served as control. Prior toinjection, the venom solution was filtered through0.22 mm Millipore filters. The volumes of both hindpaws were measured by plethysmometry (model7140 plethysmometer, Ugo Basile, Italy) before and0.25, 0.5, 1, 3, 6 and 24 h after venom administra-tion (Van Arman et al., 1965). Oedema wasexpressed as the percentage increase in the volumeof the treated (right) paw relative to that of thecontrol (left) paw at each time interval.

2.4. Effects of inhibitors of prostanoid synthesis on

BaV-and BjV-induced oedema

Various drugs that can interfere with the synthesisof prostanoids were tested. Groups of mice werepre-treated with one of the following drugs prior tothe injection of venom: (1) dexamethasone (whichcan suppress phospholipase A2 activity), 2mg/kgi.v. 2 h before venom; (2) indomethacin (a non-selective inhibitor of COX-1 and COX-2), 2mg/kgi.v. 30min before venom; (3) rofecoxib (a selectiveinhibitor of COX-2), 10mg/kg i.p. 5 and 1 h beforethe venoms; and (4) SC-560 (a selective COX-1inhibitor), 30mg/kg orally 1 h before venom. Alldrugs except indomethacin were dissolved in sterilesaline. Indomethacin was initially dissolved in 1MTris–HCl buffer (pH 8.0), then diluted with saline toprovide the desired concentration. The doses of the

ARTICLE IN PRESSR.A. Olivo et al. / Toxicon 49 (2007) 670–677672

drugs used were chosen from relevant publishedreports of their activities (Smith et al., 1998;Barbosa et al., 2003).

2.5. Paw sample preparation and prostaglandin

extraction

PGE2 extraction from paw tissue was assayedaccording to Anderson et al. (1996). Briefly, aftereuthanization, paws that had been injected withvenom or saline were amputated above the ankle,and snap-frozen in liquid nitrogen. Samples werestored at �70 1C until used. Frozen paw tissue washomogenized by politron in 1mL methanol andagain after the addition of 1mL of water. Thesamples were adjusted to 10% methanol with waterand allowed to precipitate on ice for 20min. Theprecipitated material was pelleted by centrifugationat 4 1C for 20min. The supernatants were applied toa C-18 Sep-Pak cartridge (Waters division ofMillipore Corporation, Millford, MA) that waspre-conditioned with methanol followed by water.After loading the supernatants, the columns weresequentially washed with water and 35% ethanol.The samples were eluted with ethanol and evapo-rated under nitrogen. Samples were reconstituted inELISA buffer for subsequent measurement of PGE2

concentration.

2.6. Quantification of eicosanoid concentrations

Concentrations of PGE2 were measured in pawtissue at 0.5, 1, 3 and 6 h after i.p. injections of BaV(2.5 mg/paw), BjV (0.75 mg/paw) or sterile saline.After extraction of eicosanoids on Sep Pak C18columns eluted with ethanol, the PGE2 levels weremeasured by ELISA (Pradelles et al., 1985).

2.7. Western blot analysis

The presence of COX-1 and COX-2 in pawtissue obtained 1, 3 and 6 h after injection of BaV(2.5mg/paw) or BjV (0.75mg paw), and in the contra-lateral paws injected with saline, was detected byWestern blot. The tissues were homogenized in lysisbuffer (0.1% Triton X-100, 50mM Pepstatin-A,0.2mM Leupeptin, 1mg/mL aprotinin, 10mgml�1

phenylmethyl sulfonyl fluoride, 50mM Tris and10mM EDTA). The samples were then centrifugedand the protein concentration of the supernatantdetermined by colourimetric assay (BioRad, Hercules,CA). Thirty microgram of protein was separated on

a 10% polyacrylamide gel and then transferred to anitrocellulose membrane. The membrane was in-cubated for 1 h with blocking buffer (20mM Tris,100mM NaCl, 0.5% Tween 20 and 5% non-fatdried milk) and then probed overnight with anti-body against COX-2 (1:500; Cayman Chemical,Ann Arbor, MI, USA), COX-1 and b-actin (1:500and 1:1000, respectively; Santa Cruz Biotechnology,CA, USA). The membrane was then incubated withan appropriate peroxidase-conjugated secondaryantibody for 1 h at room temperature. The im-munoreactive bands were visualized by using anenhanced chemiluminescence reagent (AmershamPharmacia). Densitometry was performed using aGS-710 Calibrated Imaging Densitometer (Bio-Rad) and analyzed with Quantity One software(Bio-Rad).

2.8. Chemicals

The ELISA kit for PGE2 was obtained fromCayman Chemicals Co. (Ann Arbor, MI, USA).SC-560 was provided by Boehringer-Ingelheim(Ingelheim, Germany). Rofecoxib was obtainedfrom Merck & Co., Inc. (Whitehouse Station, NJ,USA). Indomethacin, dexamethasone and all otherreagents were obtained from Sigma Chemical Co.(St. Louis, MO, USA).

2.9. Statistical analysis

Results are expressed as mean7SEM. Differ-ences among groups were analyzed by one-wayanalysis of variance (ANOVA) followed by eitherTukey’s test or Student’s t-test. Differences with anassociated probability (p value) of less than 5%were considered significant.

3. Results

3.1. Effects of inhibitors on oedema-formation

Intraplantar injection of BaV (2.5 mg/paw) andBjV (0.75 mg/paw) caused time-dependent increasesin paw volume (oedema). The peak increase in hindpaw swelling occurred at 30min and 1 h afterinjection of BjV and BaV, respectively, anddecreased gradually over the following 18–24 h(Figs. 1A, B). Pre-treating the mice with drugswhich interfere with arachidonate metabolism, suchas indomethacin (a non selective COX inhibitor),dexamethasone (which can suppress phospholipase

ARTICLE IN PRESSR.A. Olivo et al. / Toxicon 49 (2007) 670–677 673

A2 activity) and rofecoxib (a selective COX-2inhibitor), significantly inhibited the BaV- andBjV-induced oedema. SC-560, a selective inhibitorof COX-1, significantly reduced oedema formationinduced by BjV, but had no effect on that inducedby BaV.

0

25

50

75

100

144018060 36015

Time (min)

BaV+ Dexamethasone

BaV+ Rofecoxib

BaV

BaV+ Indomethacin

*

*

BaV+ SC-560

*

*

*

Edem

a (%

)

0

20

40

60

80

15 60 180 360 1440

Time (min)

BjV+ Dexamethasone

BjV+ Rofecoxib

BjV

BjV+ Indomethacin

* *

*

*

BjV+ SC-560

*

Edem

a (%

)

A

B

Fig. 1. Effect of inhibitors of PLA2 and cyclooxygenase on

oedema induced by Bothrops asper or Bothrops jararaca venoms.

(A) B. asper and (B) B. jararaca. Mice were pretreated with the

following drugs: dexamethasone (2mg/kg i.v. 2 h before),

indomethacin (1mg/kg i.v. 30min before); SC-560 (30mg/kg

p.o. 1 h before) and rofecoxib (10mg/kg i.p. 5 h and 1 h before

venoms); sterile saline in similar volumes and vias (control).

Then, after drug administration in the indicated time, mice

were injected in the right footpad with 2.5mg/paw of BaV or

0.75mg/paw of BjV and with saline in the left footpad. Oedema

was evaluated plethysmographically at various time intervals

after venom injection and expressed as the percentage increase in

the volume of the right footpad as compared to the left footpad.

Results are presented as mean7SEM (n ¼ 6). �Po0:05.

3.2. PGE2 synthesis by paw tissue

Concentrations of PGE2 in the paw tissue werequantified after injection of BaV (2.5 mg/paw) orBjV (0.75 mg/paw). Both venoms induced a signifi-cant increase in PGE2 levels in the paw tissuebetween 30min and 1 h after injection, as com-pared to the control paws injected with sterile saline(Fig. 2).

3.3. COX-2 and COX-1 expression

To further explore the mechanism involved invenom-induced oedema formation, the expressionof COX-1 and COX-2, was examined by Westernblotting. BaV increased COX-1 expression at 1 and3 h after injection, while BjV only caused an increaseat 3 h (Fig. 3). COX-2 expression was significantlyincreased by both venoms at 1 and 3 h afterinjection (Fig. 4).

4. Discussion

Increased vascular permeability and oedema areamong the earliest events in the inflammatoryresponse followed by infiltration of leukocytes(Vane, 1993). These events are initiated and main-tained by a sequence of inflammatory mediators ofcellular and plasma origin (Vane, 1993). After thischange, many other mechanisms are activated,contributing to the amplification of the inflamma-tory response and tissue damage. In this study, we

Control

BaV

BjV

0

50

100

150

200

30 min 1 h 3 h 6 h

*

* * *

PG

E2 (

pg/m

l)

Fig. 2. PGE2 concentration in the paw tissue after Bothrops asper

or Bothrops jararaca venom injection. Groups of mice were

injected in the paw with BaV (2.5mg/paw) or BjV (0.75mg/paw)or sterile saline (control). Concentration of PGE2 was evaluated

by specific EIA in paw tissues collected 30min, 1, 3 and 6 h after

venom injections. Each bar represents the mean7SEM from five

mice. �Po0:05 compared with corresponding control.

ARTICLE IN PRESS

1 h

3 h

6 h

C BaV BjV

COX-1

70 kDa

0

1000

2000

3000

4000

Control

BaV

B jV

1h 3h 6h

**

*

CO

X-1

Expre

ssio

n

(Arb

itra

ry U

nit

s)

A

B

Fig. 3. The effect of Bothrops asper and Bothrops jararaca venom

on COX-1 expression from soft paw tissue. The soft tissues of the

paws were collected at different times after BaV (2.5mg/paw) orBjV (0.75mg/paw) or saline injection. Tissue lysates were

obtained and subjected to electrophoresis and Western blot

analysis with an anti-COX-1 monoclonal antibody, as described

in Section 2. (A) Representative blot from three independent

experiments. (B) Densitometric analysis of immunoreactive band

intensities. Results as expressed as mean7SEM from three

experiments. �Po0:05 compared to control.

1 h

3 h

6 h

C BaV BjV

COX-2

72 kDa

Control

BaV

BjV

0

1000

2000

3000 * *

* *

1 h 3 h 6 h

CO

X-2

expre

ssio

n

(Arb

itra

ty U

nit

s)

A

B

Fig. 4. The effect of Bothrops asper and Bothrops jararaca venom

on COX-2 expression by soft paw tissue. The soft tissues of the

paws were collected at different times after BaV (2.5mg/paw) orBjV (0.75mg/paw) or saline injection. Tissue lysates were

obtained and subjected to eletrophoresis and Western blot

analysis with an anti-COX-2 polyclonal antibody, as described

in Section 2. (A) Representative blot from three independent

experiments. (B) Densitometric analysis of immunoreactive band

intensities. Results as expressed as mean7SEM from three

experiments. �Po0:05 compared to control.

R.A. Olivo et al. / Toxicon 49 (2007) 670–677674

analyzed the ability of BaV and BjV to induceoedema formation. In viper bites, oedema is theresult of the synthesis of potent autacoids oreicosanoids triggered by enzymatic components ofthe venom, as well as by the damage in themicrovasculature with plasma extravasation andthe release of cytokines (Nunez et al., 2004). Ourresults demonstrate that, in mice, BaV and BjVproduced increases in oedema formation of rapidonset and similar time-course, with maximal re-sponses between 30min and 1 h. Inhibitors of thearachidonic acid metabolism such as COX inhibi-tors and corticosteroids have inhibitory actions onoedema formation induced by numerous phlogisticagents (Perretti et al., 1996; Bressan et al., 2003). Inthe present study, drugs which interfered with themetabolism of arachidonic acid, including thecorticosteroid dexamethasone, non-selective COXinhibitor indomethacin, and rofecoxib, a selective

inhibitor of COX-2, significantly reduced theoedema formation elicited by both venoms. Thefinding that inhibitors of COX markedly attenuatedthe oedema indicated the involvement of arachido-nic acid metabolites (likely PGs) in the responses toBaV and BjV. On the other hand, a selectiveinhibitor of COX-1 (SC-560) reduced the oedemaformation induced by BjV but had no effect on thatinduced by BaV. This suggests that the mechanismsresponsible for oedema formation differ betweenthese two venoms. The present results are inagreement with others from the literature showingparticipation of COX products in the oedemainduced in rats by B. jararaca (Trebien and Calixto,1989), B. asper (Chaves et al., 1995) and B. insularis

(Barbosa et al., 2003) venoms. In contrast, theoedema produced in rats by B. lanceolatus (de Fariaet al., 2001) was reported to involve lipoxy-genase rather than COX products. Lipoxygenaseproducts have also been implicated in B. jararaca

ARTICLE IN PRESSR.A. Olivo et al. / Toxicon 49 (2007) 670–677 675

venom-induced oedema in rats (Trebien andCalixto, 1989), but are apparently not involved inmice (at least not leukotrienes C4 and D4) (Peraleset al., 1992). Also, Chacur et al. (2001) showed thatPGs do not play a significant role in the oedemaevoked by BaV in rats. Thus, these results suggestthat the mechanism(s) through which this venominduces paw oedema depend on the animal speciesstudied.

PGE2 is produced by many cells of the bodyincluding fibroblasts, macrophages, neutrophils andsome types of malignant cells (Harris et al., 2002).PGE2 is a potent mediator involved in inflammationand pain. It causes hyperalgesia, vasodilatation, andin combination with other agents, extravasation offluids to adjacent tissue (promoting oedema forma-tion) (James et al., 2001). In an attempt to under-stand the pharmacological mechanisms involved inBaV and BjV-induced inflammation in mice, theability of the venoms to stimulate PGE2 generationby paw tissue was analyzed. Intraplantar injectionsof either venom induced a significant increase inpaw tissue concentrations of PGE2 that persisted for1–3 h. These results suggest that the PGE2 releasedby BaV and BjV may contribute to the increase invascular permeability leading to oedema formation,probably synergizing with other mediators, such ashistamine and bradykinin. These data corroborateprevious studies showing the ability of Bothrops

venom to induce eicosanoid release (Farsky et al.,1997; Mello et al., 2002; Carneiro et al., 2002).

The rate-limiting enzyme for PG synthesis isCOX, which converts arachidonic acid to prosta-glandin H2. Several downstream enzymes can thenconvert PGH2 to other prostanoids, e.g., PGE2,PGI2, TXA2. There are at least two isoforms ofCOX. COX-1 is constitutively expressed in a varietyof tissues. COX-2 is not detectable in most normaltissues, but is a highly inducible gene that isexpressed in response to various proinflammatoryagents (Hegazi et al., 2003). To further analyze themechanisms involved in oedema formation, subse-quent to venom administration, the expression ofCOX-1 and COX-2 in paw tissue was analyzed. Ourresults showed that both venoms induced theexpression of COX-2 and increased the expressionof COX-1 in paw tissue. In contrast, we demon-strated that COX-1 is not expressed from peritonealleukocytes of mice after BjV injection (Moreiraet al., 2006, in press). Such discrepancy indicatesthat differences in tissues can determine differencesin the venom access to the signaling pathway

involved in the up-regulation of COXs. Our resultsshowing the increase of COX-1 expression in pawtissue after BaV and BjV injection is in agreementwith Toriyabe et al. (2004) showing increasedexpression of COX-1 in inflammatory conditions.Also, 12-O-tetradecanoyl-phorbol-13-acetate, a po-tent inflammatory agent, induces COX-1 expressionin human umbilical vein endothelial cells (Hla andNeilson, 1992) and in human monocytic leukemiaTHP-1 cells (Smith et al., 1993).

The peak of oedema formation and PGE2 releasecorrelated with marked expression of COX-2, andto a lesser extent COX-1 expression in the pawtissue. Indeed, rofecoxib, which selectively inhibitsCOX-2, markedly decreased the oedema formationinduced by either BaV or BjV. However, SC-560, aselective inhibitor of COX-1, was ineffective inaltering the changes induced by BaV. These resultshighlight a mechanistic difference in the pro-inflammatory effects of the two venoms.

Differences in the mechanism of action of thevenoms may be related to differences in their toxinsactivities. For instance, phospholipase A2 myotox-ins, isolated from BaV, are directly and indirectlyimplicated in the formation of oedema induced bythis venom (Lomonte et al., 1993; Chaves et al.,1998), and phospholipases A2 comprise approxi-mately 30% of B. asper crude venom (Gutierrez andLomonte, 1995). Considering that phospholipasesA2 are the key enzymes involved in the initiation ofthe entire eicosanoid cascade (Serhan, 1994), it ispossible that venom phospholipase A2 may play arelevant role in the expression of COX seen afterBaV injection. In contrast, BjV does not containsignificant amounts of phospholipase A2 myotoxins(Moura-da-Silva et al., 1990), and metalloprotei-nases appear to be the major components involvedin this venom-induced inflammatory events (Daleet al., 2004; Silva et al., 2004; Costa et al., 2002).Therefore, further studies are needed to investigatethis hypothesis.

In conclusion, both Bothrops venoms were able toinduce oedema formation. This event is mediated, atleast partially, by products of COX. The presentstudy provides the first experimental evidenceshowing that BaV and BjV induce the expressionof COX-1 and COX-2 in the paw tissue of mice.COX-1-derived PGs play an important role in theoedema induced by BjV, but not that induced byBaV. The use of anti-inflammatory drugs that blockCOX activity may have clinical benefit in thetreatment of patients bitten by Bothrops snakes.

ARTICLE IN PRESSR.A. Olivo et al. / Toxicon 49 (2007) 670–677676

Acknowledgments

This work was supported by FAPESP (Grantnos. 98/0162-9 and 02/13863-2). SRZ is supportedby a Fellowship from the Canadian Institutes ofHealth Research (CIHR), Janssen Pharmaceuticaand the Canadian Association of Gastroenterology.RAO is a recipient of MD fellowship from FAPESP(Grant 02/10861-9) and CFPT is a recipient ofCNPq grant.

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