Calloselasma rhodostoma is an endemic pit viper species in South East Asia. Severe local effects including swelling, blistering, compartment syndrome and tissue necrosis are commonly observed following envenoming by C. rhodostoma in Thailand15. In addition, coagulopathy resulting in hemorrhage is a major systemic outcome of C. rhodostoma envenomed patients14. C. rhodostoma envenoming-induced acute kidney injury and cardiovascular events i.e. congestive heart failure were also reported in a patient with long length of hospital in Southern Thailand14. These have raised our interest to investigate the pathological effects behind nephrotoxicity and cardiovascular disturbances using histopathological analysis. In this study the effectiveness of HPAV to inhibit morphological changes following C. rhodostoma envenoming was also investigated.
Administration of monospecific antivenom remains an effective treatment for viper envenomings. However, the availability of, and access to, geographically-appropriate antivenom, and correct identification of the biting species, remains problematic in many rural areas. Administration of polyvalent antivenom is a valid option for snakebite patients in order to minimize the occurrence of incorrect antivenom application due to diagnostic error. Previously, a number of studies exhibited the effectiveness of hemato polyvalent antivenom (HPAV) from Thailand to inhibit toxicity from various Asian hematotoxic and nephrotoxic snakes i.e. Daboia spp., Trimeresurus spp., Challoselasma spp., including Hypnale spp.16,17,19. HPAV was demonstrated to effectively neutralise procoagulant and hemorrhagic activities of all venoms tested. Moreover, HPAV also displayed a preventive effect on the occurrence of Daboia siamensis venom-induced hematuria and proteinuria in envenomed animals17. Interestingly, the potency of HPAV was shown to be generally higher than that of C. rhodostoma monovalent antivenom in neutralization of lethality, coagulation, hemorrhage and necrosis of challenge dose (5xLD50) of C. rhodostoma venom suggesting the presence of higher antibodies and synergistic cross-neutralizing component of HPAV17. Similarly, a previous study also indicated that early administration of higher concentrations of Daboia siamensis monovalent antivenom than the recommended titre (i.e. 1 ml of antvenom for 0.6 mg Daboia siamensis venom) was required to prevent nephrotoxicity following Russell’s viper envenoming19. In the present study, administration of HPAV at the recommended concentration (1 ml of antivenom to neutralise 1.6 mg Malayan pit viper venom) displayed neutralizing activity on histopathological changes of heart, liver and kidney tissues either 15 min prior to or 15 min after administration of C. rhodostoma venom (3xLD50) in mice. In a preliminary study, we found that intraperitoneal administration of C. rhodostoma venom (3xLD50) was lethal in animals tested within 1 h. Therefore, administration of HPAV 15 min after C. rhodostoma envenoming was chosen as a suitable time point to examine the effectiveness of HPAV after envenoming. There was no remarkable different in the protective effects of HPAV on envenomed mice when the antivenom was administered either 15 min prior to or after envenoming.
C. rhodostoma venom is a rich source of biological proteins such as snake venom metalloproteinase (SVMPs), phosphodiesterase (PDEs), phospholipase A2 (PLA2s), and snake venom serine protease (SVSPs). Recently, aminopeptidase, glutaminyl-peptide cyclotransferase along with ankyrin repeat were identified in Malaysian C. rhodostoma venom8. These toxic proteins were shown to be responsible for a number of hematologic outcomes (e.g. hemorrhage, hypotension and inflammation)25,26 and cellular necrosis27,28, which may involve nephrotoxicity, hepatotoxicity and cardiovasrcular disturbances observed following C. rhodostoma envenoming.
Cardiovascular effects observed following snakebite envenoming have been reported in envenomed victims of snakes from the family Elapidae (i.e. Pseudonaja textilis, Oxyuranus scutellatus and Bungarus candidus) and Viperidae (i.e. Echis ocellatus). In this study, intravenous administration of 500 µg/kg of C. rhodostoma venom caused a rapid, but transient, decrease in blood pressure and heart rate follow by a more prolonged hypotensive effect for a few minutes. In contrast, while administration of 1000 µg/kg of C. rhodostoma venom also lowered mean arterial pressure and heart rate, this was followed by cardiovascular collapse. The mechanism behind cardiovascular collapse following snakebite envenoming has been demonstrated to involve vascular mediators (e.g. nitric oxide and prostacyclin) and autonomic adaptation30,31. Previously we reported, OSC3, an isolated PLA2 from Oxyuranus scutellatus (Taipan) venom, induced a transient decrease in MAP in anaesthetized rats and caused vascular relaxation in mesenteric arteries, which was due to a combination of release of dilator autacoids and direct relaxation of vascular smooth muscle involving the cAMP/protein kinase A cascade31,32. Previously, we have shown that prior administration of hexamethonium or atropine significantly attenuated cardiac toxicity of Malayan krait (B. candidus) venom 30 suggesting the involvement of ganglionic nicotinic receptors and muscarinic acetylcholine receptors. Moreover, administration of a prothrombin activator from P. textilis venom induced cardiovascular collapse of anaesthetized rats suggesting that prothrombin activator-like toxin may be a contributor to snake venom-induced rapid cardiovascular collapse33. Indeed, acute coronary syndrome was reported in a C. rhodostoma envenomed patient, which was relieved by administration of antithrombotic agents for 5 days15. In our current study, C. rhodostoma venom caused swelling in the vasculature of cardiac muscle and the presence of macrophages in cardiac vessels indicating a direct effect of venom on tissues. The histopathological examination of heart tissue indicated that C. rhodostoma venom caused extensive hypertrophy of cardiac myofibers and mitochondrial swelling within 24 h after envenoming. These morphological changes in cardiac tissues can be attributed to the presence of cellular cytotoxic components of venom.
In this work, the effect of C. rhodostoma venom on histopathology of liver tissue was also investigated. Hepatocyte vacuolation, prominent van Kupffer cells and congestion in central vein were detected in the liver. We also detected the presence of lymphocytes pyknotic nuclei and eosinophilic cytoplasm causing amyloidosis in some areas. This indicates the presence of inflammatory effect on hepatic tissue. In fact, these hepatic injuries were also observed following Naja haje34 and Crotalus durissus terrificus35 envenoming. These hepatotoxic effects included an elevation in bilirubin, increases in serum alanine, aminotransferase, aspartate aminotransferase, γ-glutamyl transferase and alkaline phosphatase. The mechanism behind snake venom induced-hepatotoxicity was demonstrated to be associated with liver apoptosis indicated by the rise in lipid peroxidation and nitric oxide production34. In fact, snake venom L-amino acid oxidase (LAAOs) becomes an important cytotoxic agent causing cell death in several organisms via the release of reactive oxygen species, hydrogen peroxide (H2O2)36.
There are 5 groups of Asian snakes which have been reported to cause nephrotoxicity i.e. Russell’s vipers, green pit vipers, saw-scaled viper, hump-nosed pit viper and sea-snake. In the present work, we demonstrated the nephrotoxicity induced by C. rhodostoma venom. In Thailand, acute kidney injury and rhabdomyolysis (2 patients) were clinically reported following C. rhodostoma envenoming. Of these patients. one patient died from rhabdomyolysis following recovery from systemic bleeding15. Nephrotoxicity is commonly induced by snakes with hemotoxic and myotoxic effects e.g. vipers, Australian elapids and sea snakes. Clinical manifestations of renal involvement include proteinuria, hematuria, pigmenturia and acute kidney injury37. We previously demonstrated that Asian Russell’s viper (Daboia spp.) venoms contain nephrotoxic substances e.g. SVPLA2 and SVMP causing glomerulonephritis, interstitial congestion, tubular necrosis and cortical necrosis in envenomed tissues38. The present study showed that C. rhodostoma venom caused tubular necrosis with cytoplasmic eosinophilia and pyknotic nuclei, indicating the presence of inflammation of the renal tubule, similar to nephrotoxic lesions induced by other species. To treat and prevent nephrotoxicity, apart from administration of snake antivenom, early plasmapheresis and blood exchange have been applied when snake antivenom was unavailable. Moreover, plasmapheresis, blood exchange, peritoneal or hemodialysis were addressed to perform as early as possible for prevention of AKI. In addition, urine alkalization by sodium bicarbonate also help to prevent AKI in patient who has myoglobinuria or hemoglobinuria37.
In this study, we have demonstrated that C. rhodostoma envenoming causes profound histopathological changes in heart, liver and kidney tissues. Further pharmacological and physiological determinations may enable a better understanding and management of Malayan pit viper envenoming. These data also indicate that the morphological anomalies observed in envenomed tissues may contribute to cardiovascular disturbances and nephrotoxicity in envenomed victims. Early monitoring of cardiovascular and renal function together with appropriate snake antivenom administration are required to prevent the life-threatening outcomes.