J. Pineal Res. 2014; 56:295–312
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/jpi.12123
Journal of Pineal Research
Melatonin alleviates Echis carinatus venom-induced toxicities by modulating inflammatory mediators and oxidative stress Abstract: Viper bites cause high morbidity and mortality worldwide and regarded as a neglected tropical disease affecting a large healthy population. Classical antivenom therapy has appreciably reduced the snakebite mortality rate; it apparently fails to tackle viper venom-induced local manifestations that persist even after the administration of antivenom. Recently, viper venominduced oxidative stress and vital organ damage is deemed as yet another reason for concern; these are considered as postmedicated complications of viper bite. Thus, treating viper bite has become a challenge demanding new treatment strategies, auxiliary to antivenin therapy. In the last decade, several studies have reported the use of plant products and clinically approved drugs to neutralize venom-induced pharmacology. However, very few attempts were undertaken to study oxidative stress and vital organ damage. Based on this background, the present study evaluated the protective efficacy of melatonin in Echis carinatus (EC) venom-induced tissue necrosis, oxidative stress, and organ toxicity. The results demonstrated that melatonin efficiently alleviated EC venom-induced hemorrhage and myonecrosis. It also mitigated the altered levels of inflammatory mediators and oxidative stress markers of blood components in liver and kidney homogenates, and documented renal and hepatoprotective action of melatonin. The histopathology of skin, muscle, liver, and kidney tissues further substantiated the overall protection offered by melatonin against viper bite toxicities. Besides the inability of antivenoms to block local effects and the fact that melatonin is already a widely used drug promulgating a multitude of therapeutic functionalities, its use in viper bite management is of high interest and should be seriously considered.
Introduction Treating snakebite has been a major challenge considering species, geographical and seasonal variations in the venom composition [1, 2]. Thus, snake envenomation has been regarded as a major public health hazard and impacts large populations in tropical and subtropical countries including Africa, Asia, Oceania, and Latin America [3]. Viper bite management is considered critical due to the severe local and systemic tissue damage. Viper venom toxicity accounts for thousands of actual deaths and millions of cases of morbidity each year worldwide. Echis carinatus (EC), commonly known as saw scaled viper, is responsible for the highest mortality in Indian subcontinent, which is known for its severe local toxicity leading to extensive hemorrhage, edema, ecchymoses, and blister formation and causing ischemia and tissue necrosis that persist even after the administration of antivenom [4, 5]. Given the complications of viper bite treatment with classical antivenom therapy, the study demands new strategies. Now oxidative stress and vital organ damage is deemed as another major concern and are actually considered as secondary complications of viper bite [1, 6, 7]. The local tissue damage in turn triggers the production of a tremendous amount of reactive oxygen/nitrogen species
G. D. Katkar1*, M. Shanmuga Sundaram1*, M. Hemshekhar1, D. Rachana Sharma1, M. Sebastin Santhosh1, K. Sunitha1, K. S. Rangappa2, K. S. Girish1,3 and K. Kemparaju1 1
Department of Studies in Biochemistry, University of Mysore, Mysore, India; 2 Department of Studies in Chemistry, University of Mysore, Mysore, India; 3Department of Studies and Research in Biochemistry, Tumkur University, Tumkur, India Key words: Echis carinatus, hemorrhage, melatonin, oxidative damage, snake venom metalloproteases, viper venom Address reprint requests to K. Kemparaju, Professor, Department of Studies in Biochemistry, University of Mysore, Mysore-570 006, Karnataka, India. E-mail: [email protected] and K. S. Girish, Associate Professor, DOSR in Biochemistry, Tumkur University, Tumkur-572 103, Karnataka, India. E-mail: [email protected] *These authors have contributed equally to this work. Received December 27, 2013; Accepted January 31, 2014.
(ROS/RNS), key players responsible for oxidative damage, which also induce capillary perfusion resulting in cell lysis. The lysed blood cells release hemoglobin into the plasma, which relinquishes its vasculotoxic potential by directly impairing endothelial function and triggering inflammation and oxidative stress [8]. In addition, the sequel of systemic toxicities (nephrotoxicity, liver toxicity, neurotoxicity, hemostasis, and intracerebral hemorrhage) also contributes to ROS/RNS generation, thus intensifying oxidative stress and organ failure [1, 9–14]. The persistence of oxidative stress is known to damage vital organs like liver and kidney, subsequently resulting in multiple organ failure. Previously, we have demonstrated viper venominduced local toxicity, oxidative stress, and subsequent organ damage in vivo [6]. Of late, studies also have demonstrated oxidative stress-mediated hepatic and renal damage induced by Crotalus durissus terrificus (Pit viper) venom [10]. Regrettably, the classic antivenom therapy fights venom-induced systemic toxicity effectively but offers no protection against associated oxidative stress. The oxidative damage and infiltration of inflammatory mediators persist even beyond antivenom administration as a postmedicated risk. Hence, to overcome the limitations of antivenoms, an auxiliary therapy is in demand to manage 295
Katkar et al. secondary as well as post-therapeutic complications of viper envenomation. Thus, the present study was designed to verify oxidative stress and organ toxicity induced by EC venom and to test its potential inhibition by melatonin. Melatonin (N-acetyl-5-methoxytryptamine) is a hormone produced in all vertebrates including the humans and also in plants, insects, and microbes [15–17]. Physiologically, it is concerned with circadian and seasonal rhythms. It is also known to regulate cerebrovascular, reproductive, visual, neuroendocrine, and neuroimmunological functions [18, 19]. The various other functions of melatonin include immunomodulatory, anti-inflammatory, antioxidative, anxiolytic, antidepressive, sedative, and analgesic are reported [20–23]. In addition, various studies have reported the use of melatonin against neurodegenerative disorders, diabetes, hepatotoxicity, cancer, and radiation-induced complications [24–27]. Melatonin and its metabolites are efficient radical scavengers, and they have pleiotropic actions on various pathways involved in inflammation and oxidative stress [28–30]. Based on this background, the present study evaluated the inhibitory role of melatonin in EC venom-induced local and systemic toxicities, oxidative stress, and organ damage. The results demonstrate that melatonin is an effective and reliable agent to prevent viper bite-mediated local tissue degradation and subsequent oxidative stress and organ toxicity. The results were particularly noteworthy when melatonin was administered along with classical antivenom therapy.
and housed under a controlled environment. All experiments were approved by the Institutional Animal Ethical Committee (UOM/IAEC/09/2013 and UOM/IAEC/06/ 2011), Department of Studies in Zoology, University of Mysore, Mysore, and were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The experiment was divided into two parts: first was the neutralization of EC venom-induced local toxicity by melatonin (experiments were performed in mice) and second was the neutralization of EC venom-induced oxidative stress by melatonin (experiments were performed in rats). Melatonin was dissolved in absolute ethanol, and further dilutions were made in phosphate-buffered saline (PBS)/ saline. The final concentration of ethanol was
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/jpi.12123
Journal of Pineal Research
Melatonin alleviates Echis carinatus venom-induced toxicities by modulating inflammatory mediators and oxidative stress Abstract: Viper bites cause high morbidity and mortality worldwide and regarded as a neglected tropical disease affecting a large healthy population. Classical antivenom therapy has appreciably reduced the snakebite mortality rate; it apparently fails to tackle viper venom-induced local manifestations that persist even after the administration of antivenom. Recently, viper venominduced oxidative stress and vital organ damage is deemed as yet another reason for concern; these are considered as postmedicated complications of viper bite. Thus, treating viper bite has become a challenge demanding new treatment strategies, auxiliary to antivenin therapy. In the last decade, several studies have reported the use of plant products and clinically approved drugs to neutralize venom-induced pharmacology. However, very few attempts were undertaken to study oxidative stress and vital organ damage. Based on this background, the present study evaluated the protective efficacy of melatonin in Echis carinatus (EC) venom-induced tissue necrosis, oxidative stress, and organ toxicity. The results demonstrated that melatonin efficiently alleviated EC venom-induced hemorrhage and myonecrosis. It also mitigated the altered levels of inflammatory mediators and oxidative stress markers of blood components in liver and kidney homogenates, and documented renal and hepatoprotective action of melatonin. The histopathology of skin, muscle, liver, and kidney tissues further substantiated the overall protection offered by melatonin against viper bite toxicities. Besides the inability of antivenoms to block local effects and the fact that melatonin is already a widely used drug promulgating a multitude of therapeutic functionalities, its use in viper bite management is of high interest and should be seriously considered.
Introduction Treating snakebite has been a major challenge considering species, geographical and seasonal variations in the venom composition [1, 2]. Thus, snake envenomation has been regarded as a major public health hazard and impacts large populations in tropical and subtropical countries including Africa, Asia, Oceania, and Latin America [3]. Viper bite management is considered critical due to the severe local and systemic tissue damage. Viper venom toxicity accounts for thousands of actual deaths and millions of cases of morbidity each year worldwide. Echis carinatus (EC), commonly known as saw scaled viper, is responsible for the highest mortality in Indian subcontinent, which is known for its severe local toxicity leading to extensive hemorrhage, edema, ecchymoses, and blister formation and causing ischemia and tissue necrosis that persist even after the administration of antivenom [4, 5]. Given the complications of viper bite treatment with classical antivenom therapy, the study demands new strategies. Now oxidative stress and vital organ damage is deemed as another major concern and are actually considered as secondary complications of viper bite [1, 6, 7]. The local tissue damage in turn triggers the production of a tremendous amount of reactive oxygen/nitrogen species
G. D. Katkar1*, M. Shanmuga Sundaram1*, M. Hemshekhar1, D. Rachana Sharma1, M. Sebastin Santhosh1, K. Sunitha1, K. S. Rangappa2, K. S. Girish1,3 and K. Kemparaju1 1
Department of Studies in Biochemistry, University of Mysore, Mysore, India; 2 Department of Studies in Chemistry, University of Mysore, Mysore, India; 3Department of Studies and Research in Biochemistry, Tumkur University, Tumkur, India Key words: Echis carinatus, hemorrhage, melatonin, oxidative damage, snake venom metalloproteases, viper venom Address reprint requests to K. Kemparaju, Professor, Department of Studies in Biochemistry, University of Mysore, Mysore-570 006, Karnataka, India. E-mail: [email protected] and K. S. Girish, Associate Professor, DOSR in Biochemistry, Tumkur University, Tumkur-572 103, Karnataka, India. E-mail: [email protected] *These authors have contributed equally to this work. Received December 27, 2013; Accepted January 31, 2014.
(ROS/RNS), key players responsible for oxidative damage, which also induce capillary perfusion resulting in cell lysis. The lysed blood cells release hemoglobin into the plasma, which relinquishes its vasculotoxic potential by directly impairing endothelial function and triggering inflammation and oxidative stress [8]. In addition, the sequel of systemic toxicities (nephrotoxicity, liver toxicity, neurotoxicity, hemostasis, and intracerebral hemorrhage) also contributes to ROS/RNS generation, thus intensifying oxidative stress and organ failure [1, 9–14]. The persistence of oxidative stress is known to damage vital organs like liver and kidney, subsequently resulting in multiple organ failure. Previously, we have demonstrated viper venominduced local toxicity, oxidative stress, and subsequent organ damage in vivo [6]. Of late, studies also have demonstrated oxidative stress-mediated hepatic and renal damage induced by Crotalus durissus terrificus (Pit viper) venom [10]. Regrettably, the classic antivenom therapy fights venom-induced systemic toxicity effectively but offers no protection against associated oxidative stress. The oxidative damage and infiltration of inflammatory mediators persist even beyond antivenom administration as a postmedicated risk. Hence, to overcome the limitations of antivenoms, an auxiliary therapy is in demand to manage 295
Katkar et al. secondary as well as post-therapeutic complications of viper envenomation. Thus, the present study was designed to verify oxidative stress and organ toxicity induced by EC venom and to test its potential inhibition by melatonin. Melatonin (N-acetyl-5-methoxytryptamine) is a hormone produced in all vertebrates including the humans and also in plants, insects, and microbes [15–17]. Physiologically, it is concerned with circadian and seasonal rhythms. It is also known to regulate cerebrovascular, reproductive, visual, neuroendocrine, and neuroimmunological functions [18, 19]. The various other functions of melatonin include immunomodulatory, anti-inflammatory, antioxidative, anxiolytic, antidepressive, sedative, and analgesic are reported [20–23]. In addition, various studies have reported the use of melatonin against neurodegenerative disorders, diabetes, hepatotoxicity, cancer, and radiation-induced complications [24–27]. Melatonin and its metabolites are efficient radical scavengers, and they have pleiotropic actions on various pathways involved in inflammation and oxidative stress [28–30]. Based on this background, the present study evaluated the inhibitory role of melatonin in EC venom-induced local and systemic toxicities, oxidative stress, and organ damage. The results demonstrate that melatonin is an effective and reliable agent to prevent viper bite-mediated local tissue degradation and subsequent oxidative stress and organ toxicity. The results were particularly noteworthy when melatonin was administered along with classical antivenom therapy.
and housed under a controlled environment. All experiments were approved by the Institutional Animal Ethical Committee (UOM/IAEC/09/2013 and UOM/IAEC/06/ 2011), Department of Studies in Zoology, University of Mysore, Mysore, and were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The experiment was divided into two parts: first was the neutralization of EC venom-induced local toxicity by melatonin (experiments were performed in mice) and second was the neutralization of EC venom-induced oxidative stress by melatonin (experiments were performed in rats). Melatonin was dissolved in absolute ethanol, and further dilutions were made in phosphate-buffered saline (PBS)/ saline. The final concentration of ethanol was
