Hump-nosed pit vipers (genus: Hypnale
) are a group of small sized pit vipers restricted to Sri Lanka and Western Ghats of South India. These snakes are the commonest cause of snake bite in Sri Lanka [1
] and also lead to medically significant envenoming in Sri Lanka [2
] and in India [3
]. Systematics of the genus Hypnale
was recently revised by Maduwage et al. [4
] and three species namely, H. hypnale
, H. nepa
were established. Each of the three species of Hypnale
show unique geographical distribution and habitat characteristics [4
Local envenoming is the commonest clinical effect among H. hypnale
bite victims and could range from mild swelling to severe gangrene of bitten site [2
]. Coagulopathy and renal failure have also been reported in many of H. hypnale
] and in one authenticated H. zara
]. Clinical reports on H. nepa
and H. zara
bites, however, are extremely rare. Concerns on frequent potentially fatal envenoming caused by H. hypnale
bites has been raised recently [2
]. Although lethality, haemorrhagic and necrotic activity and several enzymatic activities of H. hypnale
venom has been reported [8
], such information is not available on the other two venoms. Recently, Maduwage et al. [10
] showed the reverse phase high performance liquid chromatography profiles of the three Hypnale
venoms to be similar. Further, they demonstrated similar potent cytotoxic, weak procoagulant, neurotoxic, myotoxic and phospholipase A2
activities in all three Hypnale
venoms. However, no clinical or basic in vivo experimental studies are available on the venom toxicity of H. nepa
and H. zara
The present study compares the Lethality (LD50), haemorrhagic and necrotic activity of the three Hypnale venoms and describes Hypnale venom induced pathological changes in major organs of BALB/c mice.
Lethality, haemorrhagic and necrotic activity of Hypnale venoms
Median lethal doses (LD50) with 95% confidence intervals, MLD, MHD and MND of the three Hypnale venoms are given in Table . All three Hypnale venoms showed varying degrees of haemorrhagic and necrotic activities. H. hypnale venom had lowest LD50, MHD and MND values of all three venoms, followed by H. zara and then H. nepa.
Lethality, hemorrhagic and necrotic activities of the three Hypnale venoms
Hypnale venom induced pathological changes in mouse organs
Gross and histopathological changes were observed in all organs examined except the heart. Changes observed in kidney, liver, lung, brain, spleen and intestine were similar with all three Hypnale venoms.The minimum dose of each Hypnale venom that led to each histopathological change differed drastically (Table ). No macroscopic or microscopic pathological alterations were noted in any of the above organs of the control mice (Figures g, h, f, g, h and d).
Minimum doses of the three Hypnale venoms led to each observed histopathological alteration in BALB/c mouse organs
Figure 1 Histopathological changes caused by H. hypnale, H. nepa and H. zara venoms in mouse kidneys. (Note: the histopathological changes caused by the three Hypnale venoms were similar. The dose and the type of venom led to each histopathological change in (more ...)
Figure 2 Gross and histopathological changes caused by H. hypnale, H. nepa and H. zara venoms in mouse livers. (Note: the pathological changes caused by the three Hypnale venoms were similar. The dose and the type of venom led to each pathological change in representative (more ...)
Figure 3 Histopathological changes caused by H. hypnale, H. nepa and H. zara venoms in mouse lungs, brains and spleens. (Note: the histopathological changes caused by the three Hypnale venoms were similar. The dose and the type of venom led to each histopathological (more ...)
Macroscopically, congestion and petecheal haemorrhages in medulla were observed.
Microscopically, congestion of glomeruli (Figure a) and peritubular vasculature (Figure b),petecheal haemorrhages in renal medulla (Figure c),degenerative changes in tubular cells (Figure d), tubular dilatation and flattening of tubular cells with interrupted tubular brush border were observed in mice that died as early as 3
hours of envenoming. Renal tubular necrosis (Figure e) was evident by nuclear pyknosis and shedding of the tubular cells into lumen (Figure f) predominantly in the proximal tubular cells. Necrosed tubules had intact basement membranes and these were evenly distributed within cortices. Tubular necrosis was extensive in mice that died 18 to 72
hours of envenoming. Glomerular changes were mild and were mainly restricted to congestion, dilatation and presence of hyaline material in Bowman’s capsule.
In macroscopy, oedema and congestion were commonly seen. Severe congestion with mottled appearance(Figure a) was seen in some mice. Microscopically, congestion of liver sinusoids (Figure b) and centrilobular vacuolar degeneration of hepatocytes (Figure c) was consistent at low doses. Peri-portal vacuolar degeneration of hepatocytes (Figure d) was present, less commonly. Few necrosed hepatocytes with random distribution (Figure e) were present in some sections. No haemorrhages were observed macroscopically or microscopically even at the highest venom doses tested.
Macroscopically, pulmonary congestion, gross haemorrhages and petecheal haemorrhages were evident. Pulmonary congestion, oedema (Figure a) and petecheal haemorrhages (Figure b) were observed microscopically. The latter was observed, even at venom doses less than LD50 values. Inflammatory cell infiltrate predominately with lymphocytes and with few polymorphoneuclear cells in alveolar septae and peribronchial areas was observed (Figure c).
Focal areas of neuronal degeneration (Figure e) were seen in cerebral gray matter of mice envenomed with all Hypnale venoms.
Spleens were highly congested, oedematous and friable in mice that died. However, in those that survived for 7
days, gross spleenomegaly was evident, at times being about double the normal size. In microscopy, petecheal haemorrhages were seen in red pulp of spleen in few of the mice following envenoming by H. hypnale
venom. Presence of numerous small islands of darkly stained cells in red pulp of subcapsular area (Figure f), presence of numerous aggregations of megakaryocytes (Figure g) and large aggregates of haemosiderin engulfed macrophages were seen in spleens of mice, after 7
days post envenoming.
Stomach, small and large intestine
Severe congestion was noted macroscopically and microscopically, in stomach and in the small and large intestines of mice envenomed with all three Hypnale venoms.
The present study suggests that the in-vivo toxicity of the Hypnale venoms to be different among the three species. In comparison with other two venoms, markedly low LD50 value of H. hypnale venom suggests that the overall venom toxicity of this species is higher. Further, H. hypnale venom was found to be more haemorrhagic and necrotic, than the other two species. All three venoms however, had similar pathological effects on kidney, liver, lung, brain, spleen, stomach and intestines. Renal tubular necrosis with insignificant glomerular changes, degeneration of hepatocytes, pulmonary oedema and haemorrhage, patchy neuronal degeneration in cerebral grey matter and evidence of extramedullary haemopoiesis in spleen were the major histopathological findings in mice. The minimum venom doses that resulted in pathological alterations of mouse organs suggest that H. hypnale venom is more toxic to these organs than the other two venoms. This indicates a possible difference in the severity of envenoming among victims bitten by different Hypnale species.
Evenly distributed renal tubules with degeneration and necrosis, presence of intact basement membranes in severely necrosed tubules and absence of disarrayed tubular architecture suggest direct nephrotoxicity due to Hypnale
venoms. Similar observations have been reported by Gunatilake et al. [14
], using rabbit kidney slice model. With the results of this study, it could be suggested that venoms of all three Hypnale
species are capable of causing direct tubular injury. Maduwage et al. [4
] demonstrated the potent in-vitro
cytotoxic effects of three Hypnale
venoms. Various cytotoxic components in snake venom lead to direct tubular injury in kidney [15
]. Thus, such properties may have contributed to direct tubular injury in envenomed mice.
Bilateral renal cortical necrosis has been observed in Hypnale
victims previously [4
]. In addition, evidence for presence of thrombotic microangiopathy in seven of eleven Hypnale
bite victims with severe acute kidney injury was described recently [17
]. Renal histology of these patients showed multiple glomerular capillary thrombi along with necrosis of glomeruli and tubular epithelial cells. Recently, the role of thrombotic microangiopathy in causing acute renal injury in most snake bites, predominately in viperid bites was emphasized [18
]. However, none of the mouse kidneys examined in this study had evidence of the effects of thrombotic microangiopathy. In any case, the etiology and mechanisms of developing thrombotic microangiopathy in snake bite victims has yet to be unraveled [18
Severe local necrosis is a well documented feature of H. hypnale
bite victims [2
]. However, since venoms of H. nepa
and H. zara
too showed necrotic activity in this study, local necrosis is likely to be associated with the bites caused by these two species. Potent cytotoxicity and Phospholipase A2
activity has been detected in all three Hypnale
], hence such prediction could be further justified.
Compared to the present study, intraperitoneal LD50,
MHD and MND values for H. Hypnale
reported by Tan et al. [8
] were different and it is difficult to comment on this as the latter authors have not commented on the protein quantification.
Presence of haemorrhages in lungs and kidneys with doses below the respective LD50
values and absence of such in livers even with the highest respective venom doses tested might indicate organ specific haemorrhagic activity of Hypnale
venoms. Pulmonary haemorrhage has not been observed in patients bitten by hump-nosed pit vipers as yet. It is known that metalloproteinases found in the snake venom are capable of inducing the release of inflammation mediators such as cytokines, intensifying the inflammatory response [20
]. The occurrence of a large quantity of inflammatory cells in alveolar septae of mice may be due to such activity of the three venoms.
Degeneration and necrosis of hepatocytes observed in mice livers indicate hepatotoxicity in all three Hypnale
venoms. Evidence for such activity has been observed in victims of H. hypnale
] and a victim of H. zara
]. Barraviera et al. [22
] hypothesized that hepatic injury as a frequent result of Crotaline envenomings.
Peri-portal area is the first area of the hepatic lobule to be exposed to a toxin being delivered through the bloodstream via the portal vein [23
]. Compounds administered via intraperitoneal route are absorbed primarily through the portal circulation and, therefore, passes through the liver before reaching other organs [24
]. Therefore, it could be surmised that peri-portal hepatocellular degeneration observed in mice are likely to be due to the effects of the venom absorbed via portal system.
Areas of neuronal degeneration in cerebral cortices of mice resembled ischemic neuronal degeneration. However, neither evidence of haemorrhage nor of thrombosis was revealed in these sections. Hence it is difficult to comment on the cause of these changes.
Presence of numerous small islands of darkly stained erythropoietic precursor cells seen in grossly enlarged spleens of mice, 7
days following envenoming, indicate extramedullary erythropoesis in spleen. Presence of numerous aggregations of megakaryocytes in red pulp also indicates production of platelets in spleen. Thrombocytopenia and anaemia have been observed in Hypnale
victims previously [17
]. The above observation indicates a response by spleen to such haemtological changes by hyperplasia and extramedullary haemopoesis.
Although the toxicity of the three Hypnale
venoms were found to be clearly different as revealed in this study, these differences may not be much obvious in clinical situations. This is because the amount of venom injected during actual snake bite is subjected to large variations [25
]. Several factors including size, sex, age of the snake also affect the toxicity of venom as well as the injecting venom volume in snake bite [25
]. As clinical reports on H. nepa
and H. zara
envenomings are scarce [7
] for the comparison, prospective clinical studies with accurately authenticated Hypnale
bites would be helpful in correlating these findings to actual clinical situations.