Toxicon 81 (2014) 54–57
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Letter to the Editor
The analgesics morphine and tramadol do not alter the acute toxicity induced by Bothrops asper snake venom in mice Experimental Toxinology, i.e. the study of the composition and mechanisms of action of naturally-derived venoms or toxins at the experimental level, is a scientific field that involves, among diverse methodological tools, the analysis of the actions of venoms or toxins in animals, most frequently rodents or lagomorphs, but also in larger species. Administration of venoms or toxins generally causes pain. A typical example is the use of viperid snake venoms, or purified tissue-damaging toxins from these venoms. For instance, the venom of the pit viper Bothrops asper, as well as myotoxic PLA2s and PLA2 homologs, and hemorrhagic SVMPs isolated from this venom, provoke pain, i.e. hyperalgesia and allodynia, in rodents (Chacur et al., 2001, 2003, 2004a, 2004b; Teixeira et al., 2003; Fernandes et al., 2007). This is also the case with many types of animal venoms and toxins and microbial toxins which induce soft tissue necrosis and inflammation, and systemic alterations associated with pain. There is a widespread and growing concern with the suffering, specifically the generation of pain, that experimental animals undergo in tests designed to evaluate the toxicological profile and mechanism of action of venoms and toxins, and the ability of therapeutic agents, such as antivenoms, to neutralize these effects. In particular, the preclinical assessment of the neutralizing ability of antivenoms involves the injection of venoms, or of mixtures of venoms and antivenoms, to experimental animals, mostly mice, with the consequent generation of distress and pain. In addition to the gold standard for evaluating antivenom efficacy, i.e. the assay for neutralization of lethality in mice, the WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins (WHO, 2010) include various additional assays for the analysis of the preclinical efficacy of antivenoms, in order to have a more integrated view of the neutralizing profile of these immunotherapeutics. These are the neutralization of hemorrhagic, myotoxic, edema-forming, dermonecrotic and defibrinogenating activities, which characterize envenomings by viperid venoms and, in some cases, by elapid snake venoms as http://dx.doi.org/10.1016/j.toxicon.2014.02.001 0041-0101/Ó 2014 Elsevier Ltd. All rights reserved.
well (Theakston and Reid, 1983; WHO, 2010; Gutiérrez et al., 2013). The reduction of pain in these assays, as part of the general ethical framework of replacement, reduction and refinement of the laboratory methods performed in animals (Russell and Burch, 1959; Robinson, 2005) should receive more attention from the toxinological research community. The refinement, defined as any approach that reduces or eliminates the potential pain or distress in animals, and which enhances animal wellbeing, is of particular importance in toxicity testing (Holmes et al., 2010; Madden et al., 2012). One of the reasons why toxinologists have been slow in the introduction of precautionary analgesia in experimental research has to do with the concern that this intervention might affect the results of the assays. In this context, proper validation of the test method is necessary to demonstrate that the alternative proposed is scientifically valid and generates the same results (Schechtman, 2002). Recently, Harris et al. (2013) highlighted the need to perform research on the use of analgesics in experimental Toxinology. They described the experience gained in their laboratory for many years with the use of the m-selective opioid buprenorphine. In this communication we present results on the use of the analgesics morphine and tramadol in the study of hemorrhagic, myotoxic, edema-forming and defibrinogenating activities of B. asper venom in mice, and whether the use of these analgesics affects the outcome of the toxinological tests performed. In all cases, groups of CD-1 mice (18–20 g) were pretreated with either distilled water or analgesics (either morphine sulfate (Laboratorio Sanderson S.A., Chile, 5 and 10 mg/kg) or tramadol chlorhydrate (Laboratorio Sanderson S.A., Chile, 50 mg/kg) by the subcutaneous route). The doses of analgesics used were selected on the basis of previous studies estimating the analgesic effect of these drugs in mice (Kissel et al., 1961; Umans and Inturrisi, 1981; Raffa et al., 1992; Gades et al., 2000; Díaz-Reval et al., 2010). Fifteen min after administration of either analgesic or vehicle (water), animals were injected with B. asper venom
20
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Letter to the Editor / Toxicon 81 (2014) 54–57
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Fig. 1. Hemorrhagic (A), myotoxic (B), and edema-forming (C, D) activities of Bothrops asper venom in mice. Groups of mice were injected subcutaneously with either morphine (5 mg/kg), tramadol (50 mg/kg) or water, in a total volume of 100 mL. Fifteen min afterward, animals were injected with B. asper venom for the assessment of hemorrhagic, myotoxic and edema-forming activities, as described in the text. In the case of edema-forming activity, (C) shows the time-course of the effect in mice pretreated with either water or morphine, whereas (D) shows the results in mice pretreated with either water or tramadol. Results are presented as mean S.D. (n ¼ 5). No significant differences (p > 0.05) were observed in any of the effects between mice pretreated with water and those pretreated with the analgesics.
for the assessment of each effect. For hemorrhagic activity, 20 mg of venom, dissolved in 100 mL of 0.14 M NaCl, 0.04 M phosphate, pH 7.2 (PBS), were injected intradermally in the ventral abdominal region of mice (n ¼ 5). Two hr after injection, animals were sacrificed by CO2 inhalation, their skins were removed and the diameter of the hemorrhagic halo in the inner side of the skin was measured (Gutiérrez et al., 1985). For myotoxic activity, similar groups of mice (n ¼ 5) were injected intramuscularly, in the right gastrocnemius, with 50 mg venom dissolved in 50 mL PBS. After 3 h, mice were bled from the tail and the creatine kinase (CK) activity of plasma was determined using a commercial kit (CK LIQUI-UV, Stanbio Lab., Texas, USA) (Gutiérrez et al., 1980). Afterward, animals were sacrificed by CO2 inhalation, the injected gastrocnemius muscle was dissected out, and tissue samples were immersed in 10% formaldehyde fixative solution. After routine processing, samples were embedded in paraffin, and sections were stained with hematoxylin and eosin for microscopic examination. For edema-forming activity, mice (n ¼ 5) were injected subcutaneously in the right foot pad with 5 mg venom dissolved in 50 mL PBS. The thickness of the footpad was measured with a low-pressure spring caliper (Lomonte et al., 1993) before injection and at various time intervals after injection. Defibrinogenating activity was determined using the method of Theakston and Reid (1983), as
modified by Gené et al. (1989). Briefly, groups of mice (n ¼ 3) were injected i.v. with various doses of venom, dissolved in 100 mL PBS. One hr after injection, a sample of blood was collected by cardiac puncture under anesthesia with ketamine and xylazine. Two hundred mL of blood were placed in dry glass tubes and allowed to stand at room temperature for 20 min. The Minimum Defibrinogenating Dose (MDD) corresponds to the venom dose at which the blood from the three animals remained unclottable (Gené et al., 1989). In all cases, control mice were injected with the same volume of PBS without venom. Experiments were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of the University of Costa Rica. As shown in Fig. 1, there were not significant differences in the magnitude of hemorrhagic, myotoxic and edemaforming effects of B. asper venom in mice pretreated with either distilled water, or morphine or tramadol at the doses used. In agreement, histological assessment of muscle tissue revealed a similar extent of hemorrhage and myonecrosis in mice pretreated with water or analgesics and then injected with venom (not shown). Likewise, no difference was observed in the estimation of the defibrinogenating effect, since the MDD corresponded to 5 mg in animals pretreated with either water or the analgesics. Mice pretreated with the analgesics showed notoriously less distress and discomfort after envenoming than those
56
Letter to the Editor / Toxicon 81 (2014) 54–57
pretreated with water, as judged by their general behavior. Control mice injected with PBS alone did not develop hemorrhage, myonecrosis, edema or defibrinogenation nor did they have any evidence of distress or discomfort. Results clearly show that pretreatment of mice with morphine and tramadol, at doses previously described to exert analgesic effect in mice, do not affect the magnitude of the acute toxicity induced by B. asper venom in mice, i.e. hemorrhagic, myotoxic, edema-forming and defibrinogenating effects. This is in agreement with what is known on the mechanism of action of these analgesics, since they are agonists of opioid receptors (m, d and k) and act primarily at spinal and supraspinal regions involved in the transmission and modulation of pain (Pasternak, 1993; Yaksh, 1997). These analgesics may also cause other central and peripheral actions which are unrelated to the venom-induced pathological effects studied (Pasternak, 1993; Yaksh, 1997). Tramadol is a weak opioid agonist, but it is also an inhibitor of monoamine neurotransmitter reuptake, and unlike other opioids it has no effects on respiratory or cardiovascular function, and has a low potential to generate tolerance (Raffa et al., 1992; Scott and Perry, 2000). The time lapse of the analgesic effect of morphine and tramadol in mice have been estimated to be 2–3 h (Umans and Inturrisi, 1981; Gades et al., 2000; Ide et al., 2006), and the onset of action of both analgesics in mice and rats is around 15 min (Kissel et al., 1961; Umans and Inturrisi, 1981; Raffa et al., 1992). Therefore, these analgesics are useful for experiments in which toxicity is assessed within few hours after venom or toxin injection. Buprenorphine has a more prolonged duration of action, estimated to be between 6 and 12 h in the rat (Gades et al., 2000; Roughan and Flecknell, 2002) and 3 to 5 h in mice (Gades et al., 2000). The formal introduction of the use of precautionary analgesia in toxinological research should be promoted. Although extrapolation of these results to other experimental models and venoms demands caution, it is highly likely that similar results may be obtained with other viperid snake venoms, owing to similarities in the mechanisms of action of myotoxic, hemorrhagic, edemaforming and defibrinogenating toxins. Further studies with these and additional analgesics and with other venoms and toxins are needed in order to provide information that would support the preparation of guidelines for the routine use of precautionary (prophylactic) analgesia in the study of acute toxicity of venoms and toxins, and their neutralization by antivenoms as part of the general efforts to reduce suffering of animals in experimental Toxinology. Acknowledgment Thanks are due to Daniela Solano (Instituto Clodomiro Picado) for her collaboration in the experiments described in this letter. Conflict of interest statement The authors declare that there are no conflicts of interests regarding this work.
References Chacur, M., Gutiérrez, J.M., Milligan, E.D., Wieseler-Frank, J., Britto, L.R., Maier, S.F., Watkins, L.R., Cury, Y., 2004a. Snake venom components enhance pain upon subcutaneous injection: an initial examination of spinal cord mediators. Pain 111, 65–76. Chacur, M., Longo, I., Picolo, G., Gutiérrez, J.M., Lomonte, B., Guerra, J.L., Teixeira, C.F.P., Cury, Y., 2003. Hyperalgesia induced by Asp49 and Lys49 phospholipases A2 from Bothrops asper snake venom: pharmacological mediation and molecular determinants. Toxicon 41, 667– 678. Chacur, M., Milligan, E.D., Sloan, E.M., Wieseler-Frank, J., Barrientos, R.M., Martin, D., Poole, S., Lomonte, B., Gutiérrez, J.M., Maier, S.F., Cury, Y., Watkins, L., 2004b. Snake venom phospholipases A2 (Asp49 and Lys49) induce mechanical allodynia upon peri-sciatic administration: involvement of spinal cord glia, proinflammatory cytokines and nitric oxide. Pain 108, 180–191. Chacur, M., Picolo, G., Gutiérrez, J.M., Teixeira, C.F.P., Cury, Y., 2001. Pharmacological modulation of hyperalgesia induced by Bothrops asper (teriopelo) snake venom. Toxicon 39, 1173–1181. Díaz-Reval, M.I., Carrillo-Munguía, N., Martínez-Casas, M., GonzálezTrujano, M.E., 2010. Tramadol and caffeine produce synergistic interactions on antinociception measured in a formalin model. Pharmacol. Biochem. Behav. 97, 357–362. Fernandes, C.M., Teixeira, C.F.P., Leite, A.C., Gutiérrez, J.M., Rocha, F.A., 2007. The snake venom metalloproteinase BaP1 induces joint hypernociception through TNF-a and PGE2-dependent mechanisms. Br. J. Pharmacol. 151, 1254–1261. Gades, N.M., Danneman, P.J., Wixson, S.K., Tolley, E.A., 2000. The magnitude and duration of the analgesic effect of morphine, butorphanol, and buprenorphine in rats and mice. Contemp. Top. Lab. Anim. Sci. 39, 8–13. Gené, J.A., Roy, A., Rojas, G., Gutiérrez, J.M., Cerdas, L., 1989. Comparative study on coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of Costa Rican crotaline snake venoms and their neutralization by a polyvalent antivenom. Toxicon 27, 841–848. Gutiérrez, J.M., Arroyo, O., Bolaños, R., 1980. Mionecrosis, hemorragia y edema inducidos por el veneno de Bothrops asper en ratón blanco. Toxicon 18, 603–610. Gutiérrez, J.M., Gené, J.A., Rojas, G., Cerdas, L., 1985. Neutraliztion of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom. Toxicon 23, 887–893. Gutiérrez, J.M., Solano, G., Pla, D., Herrera, M., Segura, Á., Villalta, M., Vargas, M., Sanz, L., Lomonte, B., Calvete, J.J., León, G., 2013. Assessing the preclinical efficacy of antivenoms: from the lethality neutralization assay to antivenomics. Toxicon 69, 168–179. Harris, J., Flecknell, P., Thomas, A., Warrell, D.A., 2013. On the use of analgesia in experimental toxinology. Toxicon 64, 36–37. Holmes, A.M., Creton, S., Chapman, K., 2010. Working in partnership to advance the 3Rs in toxicity testing. Toxicology 267, 4–19. Ide, S., Minami, M., Ishihara, K., Uhl, G.R., Sora, I., Ikeda, K., 2006. m opioid receptor-dependent and independent components in effects of tramadol. Neuropharmacology 51, 651–658. Kissel, J.W., Albert, J.R., Boxill, G.C., 1961. The pharmacology of prodilidine hydrochloride, a new analgetic agent. J. Pharmacol. Exp. Ther. 134, 332–340. Lomonte, B., Tarkowski, A., Hanson, L.Å., 1993. Host response to Bothrops asper snake venom. Analysis of edema formation, inflammatory cells, and cytokine release in a mouse model. Inflammation 17, 93– 105. Madden, J.C., Hewitt, M., Przybylak, K., Vandebriel, R.J., Piersma, A.H., Cronin, M.T.D., 2012. Strategies for the optimisation of in vivo experiments in accordance with the 3Rs philosophy. Regul. Toxicol. Pharmacol. 63, 140–154. Pasternak, G.W., 1993. Pharmacological mechanisms of opioid analgesics. Clin. Neuropharmacol. 16, 1–18. Raffa, R.B., Friderichs, E., Reimann, W., Shank, R.P., Codd, E.E., Vaught, J.L., 1992. Opioid and nonopioid components independently contribute to the mechanism of action of tramadol, an ‘atypical’ opioid analgesic. J. Pharmacol. Exp. Ther. 260, 275–285. Robinson, V., 2005. Finding alternatives: an overview of the 3Rs and the use of animals in research. Sch. Sci. Rev. 87, 1–4. Roughan, J.V., Flecknell, P.A., 2002. Evaluation of a short-duration behaviour-based post-operative pain scoring system in rats. Eur. J. Pain 7, 397–406. Russell, W.M.S., Burch, R.L., 1959. The Principles of Humane Experimental Technique. Methuen & Co. Ltd, London. Schechtman, L.M., 2002. Implementation of the 3Rs (Refinement, reduction, and Replacement): validation and regulatory acceptance
Letter to the Editor / Toxicon 81 (2014) 54–57 considerations for alternative toxicological test methods. ILAR J. 43 (Suppl. 1), S85–S94. Scott, L.J., Perry, C.M., 2000. Tramadol. Drugs 60, 139–176. Teixeira, C.F.P., Landucci, E., Antunes, E., Chacur, M., Cury, Y., 2003. Inflammatory effects of snake venom myotoxic phospholipases A2. Toxicon 42, 947–962. Theakston, R.D.G., Reid, H.A., 1983. Development of simple standard procedures for the characterization of snake venom. Bull. World Health Organ. 61, 949–956. Umans, J.G., Inturrisi, C.E., 1981. Pharmacodynamics of subcutaneously administered diacetylmorphine, 6-acetylmorphine and morphine in mice. J. Pharmacol. Exp. Ther. 218, 409–415. World Health Organization, 2010. WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins. World Health Organization, Geneva. Available in: http://www. who.int/bloodproducts/snake_antivenoms/snakeantivenomguide/ en/. Yaksh, T.L., 1997. Pharmacology and mechanisms of opioid analgesic activity. Acta Anesthesiol. Scand. 41, 94–111.
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José María Gutiérrez* Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José 1000, Costa Rica Cristina Herrera Facultad de Farmacia, Universidad de Costa Rica, San José, Costa Rica Corresponding author. Tel.: þ506 2511 7865; fax: þ506 2292 0485. E-mail addresses: [email protected], [email protected] (J.M. Gutiérrez) 9 January 2014 Available online 13 February 2014
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Letter to the Editor
The analgesics morphine and tramadol do not alter the acute toxicity induced by Bothrops asper snake venom in mice Experimental Toxinology, i.e. the study of the composition and mechanisms of action of naturally-derived venoms or toxins at the experimental level, is a scientific field that involves, among diverse methodological tools, the analysis of the actions of venoms or toxins in animals, most frequently rodents or lagomorphs, but also in larger species. Administration of venoms or toxins generally causes pain. A typical example is the use of viperid snake venoms, or purified tissue-damaging toxins from these venoms. For instance, the venom of the pit viper Bothrops asper, as well as myotoxic PLA2s and PLA2 homologs, and hemorrhagic SVMPs isolated from this venom, provoke pain, i.e. hyperalgesia and allodynia, in rodents (Chacur et al., 2001, 2003, 2004a, 2004b; Teixeira et al., 2003; Fernandes et al., 2007). This is also the case with many types of animal venoms and toxins and microbial toxins which induce soft tissue necrosis and inflammation, and systemic alterations associated with pain. There is a widespread and growing concern with the suffering, specifically the generation of pain, that experimental animals undergo in tests designed to evaluate the toxicological profile and mechanism of action of venoms and toxins, and the ability of therapeutic agents, such as antivenoms, to neutralize these effects. In particular, the preclinical assessment of the neutralizing ability of antivenoms involves the injection of venoms, or of mixtures of venoms and antivenoms, to experimental animals, mostly mice, with the consequent generation of distress and pain. In addition to the gold standard for evaluating antivenom efficacy, i.e. the assay for neutralization of lethality in mice, the WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins (WHO, 2010) include various additional assays for the analysis of the preclinical efficacy of antivenoms, in order to have a more integrated view of the neutralizing profile of these immunotherapeutics. These are the neutralization of hemorrhagic, myotoxic, edema-forming, dermonecrotic and defibrinogenating activities, which characterize envenomings by viperid venoms and, in some cases, by elapid snake venoms as http://dx.doi.org/10.1016/j.toxicon.2014.02.001 0041-0101/Ó 2014 Elsevier Ltd. All rights reserved.
well (Theakston and Reid, 1983; WHO, 2010; Gutiérrez et al., 2013). The reduction of pain in these assays, as part of the general ethical framework of replacement, reduction and refinement of the laboratory methods performed in animals (Russell and Burch, 1959; Robinson, 2005) should receive more attention from the toxinological research community. The refinement, defined as any approach that reduces or eliminates the potential pain or distress in animals, and which enhances animal wellbeing, is of particular importance in toxicity testing (Holmes et al., 2010; Madden et al., 2012). One of the reasons why toxinologists have been slow in the introduction of precautionary analgesia in experimental research has to do with the concern that this intervention might affect the results of the assays. In this context, proper validation of the test method is necessary to demonstrate that the alternative proposed is scientifically valid and generates the same results (Schechtman, 2002). Recently, Harris et al. (2013) highlighted the need to perform research on the use of analgesics in experimental Toxinology. They described the experience gained in their laboratory for many years with the use of the m-selective opioid buprenorphine. In this communication we present results on the use of the analgesics morphine and tramadol in the study of hemorrhagic, myotoxic, edema-forming and defibrinogenating activities of B. asper venom in mice, and whether the use of these analgesics affects the outcome of the toxinological tests performed. In all cases, groups of CD-1 mice (18–20 g) were pretreated with either distilled water or analgesics (either morphine sulfate (Laboratorio Sanderson S.A., Chile, 5 and 10 mg/kg) or tramadol chlorhydrate (Laboratorio Sanderson S.A., Chile, 50 mg/kg) by the subcutaneous route). The doses of analgesics used were selected on the basis of previous studies estimating the analgesic effect of these drugs in mice (Kissel et al., 1961; Umans and Inturrisi, 1981; Raffa et al., 1992; Gades et al., 2000; Díaz-Reval et al., 2010). Fifteen min after administration of either analgesic or vehicle (water), animals were injected with B. asper venom
20
A CK (Units/L)
15 10 5
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5
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Water Tramadol
____________________________
Increment in footpad thickness (mm)
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12000
6
Time (hr)
Increment in footpad thickness (mm)
Hemorrhagic diameter (mm)
Letter to the Editor / Toxicon 81 (2014) 54–57
Venom
D
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 0
1
2
3
4
5
6
Time (hr)
Fig. 1. Hemorrhagic (A), myotoxic (B), and edema-forming (C, D) activities of Bothrops asper venom in mice. Groups of mice were injected subcutaneously with either morphine (5 mg/kg), tramadol (50 mg/kg) or water, in a total volume of 100 mL. Fifteen min afterward, animals were injected with B. asper venom for the assessment of hemorrhagic, myotoxic and edema-forming activities, as described in the text. In the case of edema-forming activity, (C) shows the time-course of the effect in mice pretreated with either water or morphine, whereas (D) shows the results in mice pretreated with either water or tramadol. Results are presented as mean S.D. (n ¼ 5). No significant differences (p > 0.05) were observed in any of the effects between mice pretreated with water and those pretreated with the analgesics.
for the assessment of each effect. For hemorrhagic activity, 20 mg of venom, dissolved in 100 mL of 0.14 M NaCl, 0.04 M phosphate, pH 7.2 (PBS), were injected intradermally in the ventral abdominal region of mice (n ¼ 5). Two hr after injection, animals were sacrificed by CO2 inhalation, their skins were removed and the diameter of the hemorrhagic halo in the inner side of the skin was measured (Gutiérrez et al., 1985). For myotoxic activity, similar groups of mice (n ¼ 5) were injected intramuscularly, in the right gastrocnemius, with 50 mg venom dissolved in 50 mL PBS. After 3 h, mice were bled from the tail and the creatine kinase (CK) activity of plasma was determined using a commercial kit (CK LIQUI-UV, Stanbio Lab., Texas, USA) (Gutiérrez et al., 1980). Afterward, animals were sacrificed by CO2 inhalation, the injected gastrocnemius muscle was dissected out, and tissue samples were immersed in 10% formaldehyde fixative solution. After routine processing, samples were embedded in paraffin, and sections were stained with hematoxylin and eosin for microscopic examination. For edema-forming activity, mice (n ¼ 5) were injected subcutaneously in the right foot pad with 5 mg venom dissolved in 50 mL PBS. The thickness of the footpad was measured with a low-pressure spring caliper (Lomonte et al., 1993) before injection and at various time intervals after injection. Defibrinogenating activity was determined using the method of Theakston and Reid (1983), as
modified by Gené et al. (1989). Briefly, groups of mice (n ¼ 3) were injected i.v. with various doses of venom, dissolved in 100 mL PBS. One hr after injection, a sample of blood was collected by cardiac puncture under anesthesia with ketamine and xylazine. Two hundred mL of blood were placed in dry glass tubes and allowed to stand at room temperature for 20 min. The Minimum Defibrinogenating Dose (MDD) corresponds to the venom dose at which the blood from the three animals remained unclottable (Gené et al., 1989). In all cases, control mice were injected with the same volume of PBS without venom. Experiments were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of the University of Costa Rica. As shown in Fig. 1, there were not significant differences in the magnitude of hemorrhagic, myotoxic and edemaforming effects of B. asper venom in mice pretreated with either distilled water, or morphine or tramadol at the doses used. In agreement, histological assessment of muscle tissue revealed a similar extent of hemorrhage and myonecrosis in mice pretreated with water or analgesics and then injected with venom (not shown). Likewise, no difference was observed in the estimation of the defibrinogenating effect, since the MDD corresponded to 5 mg in animals pretreated with either water or the analgesics. Mice pretreated with the analgesics showed notoriously less distress and discomfort after envenoming than those
56
Letter to the Editor / Toxicon 81 (2014) 54–57
pretreated with water, as judged by their general behavior. Control mice injected with PBS alone did not develop hemorrhage, myonecrosis, edema or defibrinogenation nor did they have any evidence of distress or discomfort. Results clearly show that pretreatment of mice with morphine and tramadol, at doses previously described to exert analgesic effect in mice, do not affect the magnitude of the acute toxicity induced by B. asper venom in mice, i.e. hemorrhagic, myotoxic, edema-forming and defibrinogenating effects. This is in agreement with what is known on the mechanism of action of these analgesics, since they are agonists of opioid receptors (m, d and k) and act primarily at spinal and supraspinal regions involved in the transmission and modulation of pain (Pasternak, 1993; Yaksh, 1997). These analgesics may also cause other central and peripheral actions which are unrelated to the venom-induced pathological effects studied (Pasternak, 1993; Yaksh, 1997). Tramadol is a weak opioid agonist, but it is also an inhibitor of monoamine neurotransmitter reuptake, and unlike other opioids it has no effects on respiratory or cardiovascular function, and has a low potential to generate tolerance (Raffa et al., 1992; Scott and Perry, 2000). The time lapse of the analgesic effect of morphine and tramadol in mice have been estimated to be 2–3 h (Umans and Inturrisi, 1981; Gades et al., 2000; Ide et al., 2006), and the onset of action of both analgesics in mice and rats is around 15 min (Kissel et al., 1961; Umans and Inturrisi, 1981; Raffa et al., 1992). Therefore, these analgesics are useful for experiments in which toxicity is assessed within few hours after venom or toxin injection. Buprenorphine has a more prolonged duration of action, estimated to be between 6 and 12 h in the rat (Gades et al., 2000; Roughan and Flecknell, 2002) and 3 to 5 h in mice (Gades et al., 2000). The formal introduction of the use of precautionary analgesia in toxinological research should be promoted. Although extrapolation of these results to other experimental models and venoms demands caution, it is highly likely that similar results may be obtained with other viperid snake venoms, owing to similarities in the mechanisms of action of myotoxic, hemorrhagic, edemaforming and defibrinogenating toxins. Further studies with these and additional analgesics and with other venoms and toxins are needed in order to provide information that would support the preparation of guidelines for the routine use of precautionary (prophylactic) analgesia in the study of acute toxicity of venoms and toxins, and their neutralization by antivenoms as part of the general efforts to reduce suffering of animals in experimental Toxinology. Acknowledgment Thanks are due to Daniela Solano (Instituto Clodomiro Picado) for her collaboration in the experiments described in this letter. Conflict of interest statement The authors declare that there are no conflicts of interests regarding this work.
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José María Gutiérrez* Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José 1000, Costa Rica Cristina Herrera Facultad de Farmacia, Universidad de Costa Rica, San José, Costa Rica Corresponding author. Tel.: þ506 2511 7865; fax: þ506 2292 0485. E-mail addresses: [email protected], [email protected] (J.M. Gutiérrez) 9 January 2014 Available online 13 February 2014
