H. TOPYILDIZ, S. HAYRETDAĞ

Turk J Zool 2012; 36(4): 517-525 © TÜBİTAK Research Article doi:10.3906/zoo-1007-23

Histopathological eff ects of xanthina venom on rats

Hüseyin TOPYILDIZ, Sibel HAYRETDAĞ* Department of Biology, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, 17100 Çanakkale -

Received: 16.07.2010

Abstract: In this study, the histopathological changes caused by venom to rat skin, skeletal muscle, and liver tissue were analysed by light microscope. Th e venom (approximately 2.85 mg/kg) was injected intramuscularly into the gastrocnemius muscle of the rats. Th e were dissected 1, 3, and 6 h aft er the injection time, and tissue samples were taken. Sections 5 μm thick were taken from paraffi n blocks of tissue samples, and the sections were stained with haematoxylin and eosin in order to facilitate examination by light microscope. Examinations showed no damage to the epidermis layer of skin. However, damage to the dermis layer, including oedema, haemorrhage, local bleeding, rare infl ammatory cells, strong cell infi ltration with mixed characteristics, infl ammation around the veins, and fat necrosis, was detected. Th e damage observed in muscular tissue was myonecrosis, mixed character cell infi ltration, haemorrhage, and the formation of inclusion on myofi brils. Th e liver tissue revealed sinusoidal congestion and hepatocellular degeneration.

Key words: Montivipera xanthina, histopathology, venom, skin, skeletal muscle, liver

Sıçanlarda Montivipera xanthina venomunun histopatolojik etkileri

Özet: Bu çalışmada Montivipera xanthina zehirinin, sıçanların deri, iskelet kası ve karaciğer dokularında meydana getirdiği histopatolojik değişiklikler ışık mikroskobu ile incelenmiştir. Venom (yaklaşık 2,85 mg/kg) sıçanların gastrocnemius kası içerisine enjekte edilmiştir. Enjeksiyondan 1, 3 ve 6 saat sonra hayvanlar disekte edilmiş ve doku örnekleri alınmıştır. Bu örneklerden hazırlanan parafi n bloklardan 5 mikron kalınlığında alınan kesitler hematoksilen ve eosin ile boyanarak ışık mikroskobunda incelenmiştir. İncelemeler sonucunda derinin epidermis kısmında bir hasara rastlanmamıştır. Dermis kısmında ise ödem, hemoraji, lokal kanlanmalar, seyrek iltihabi hücreler, karışık karakterli şiddetli hücre infi ltrasyonu, damar etrafı yangı ve yağ nekrozu tespit edilmiştir. Kas dokusunda miyonekroz, karışık karakterli hücre infi ltrasyonu, hemoraji, miyofi brillerde inklüzyon oluşumu gözlenen hasarlardır. Karaciğer dokusu incelendiğinde sinüzoidal kanlanma ve hepatoselüler dejenerasyon tespit edilmiştir.

Anahtar sözcükler: Montivipera xanthina, histopatoloji, venom, deri, iskelet kası, karaciğer

* E-mail: [email protected]

517 Histopathological eff ects of Montivipera xanthina venom on rats

Introduction aim of this study was to histopathologically examine Venom in is produced, stored, and secreted the eff ects of intramuscularly injected M. xanthina in venom glands (Duvernoy’s glands). Th e venom venom on rat tissues such as skin, skeletal muscle, consists of organic ingredients such as proteins, and liver tissues. glycoprotein, and phospholipids, as well as inorganic ingredients such as Ca, Cu, Fe, K, Mg, Mn, Na, P, Co, Materials and methods and Zn. Components of the venom vary between and that variation produces diff erent Montivipera xanthina and venom reactions in living organisms (Bjarnason and Fox, Venom was extracted from adolescent M. xanthina 1989). individuals from the Aegean Region in the western Th e eff ects of venom can be classifi ed into 2 part of Turkey. Th e snakes were kept in a terrarium separate groups: neurotoxins and haemolytic toxins. in the laboratory. When the venom was extracted, Some of the main biological eff ects of snake venoms the snakes were ‘secured’ on the ground by pushing are neurotoxic and cause coagulation, cytotoxic, on the spot between their heads and necks with an haemolysis, haemorrhagic activity, hypotension, and L-shaped stick, and their heads were held with bare necrosis (Bjarnason and Fox, 1989; Warrell, 1989). hands. Th e snakes’ venom teeth were sunk into a 50-mL beaker that was prepared beforehand and Leonardi et al. (2001) established that Vipera ammodytes haemorrhagin protein 1, which is covered with Parafi lm. Th e venom in the beaker was present in the venom of Vipera ammodytes, causes transferred into sterilised tubes. Th e venom in the coagulation in the blood. Another relevant study tubes was immediately centrifuged at 2850 rpm for suggests that Macrovipera lebetina venom causes 5 min through a cooling centrifuge (Arıkan et al., coagulation as well (Samel et al., 2002). In another 2003). Aft er precipitating tissues and other particles study carried out on albino rats and guinea pigs, to the bottom of the tubes, the supernatant was Vipera ammodytes venom was the cause of gangrene transferred to a sterilised Eppendorf tube. Th e clean found in muscular tissue (Balija et al., 2005). A study venom was extracted, lyophilised, and stored at −20 executed by Aznaurian and Amiryan (2006) focused °C (Acosta et al., 2003). Before application to the rats, on the damage caused by venom the venom was dissolved in 10 μL of physiological to rabbit tissues including the liver, heart, kidneys, saline solution according to dose. lungs, spleen, and adrenal gland. Results of the study Dosage showed that the venom causes serious tissue damage Th e dose given to the rats was based on a ratio of such as kidney failure due to an accumulation of 200 mg of venom with a value of LD for a human blood in the glomerulus as a result of nephrotoxic 50 body weighing 70 kg (Başoğlu and Baran, 1980). In eff ects and cell fragmentation-based gangrene in accordance with this formula, individual doses were lung tissue. calculated for the rats; their weights varied between Research has established that 42 snake species 200 and 400 g. Th e venom doses calculated for each inhabit Turkey, and 13 of these species are venomous. rat were dissolved in 10 μL of physiological saline Among the venomous species, 1 belongs to Elapidae, solution and intramuscularly injected into the right 10 are from , and the remaining 2 are gastrocnemius muscle of the rats. semivenomous (Başoğlu and Baran, 1980; Baran, Animals and venom application 2005). Th e endemic snake M. xanthina is one of the most dangerous among these species (Baran and Th e 25 male rats (albino Wistar; Rattus rattus) used Atatür, 1998). A set of studies executed by Arıkan et in this study were purchased from the Experimental al. (2003) gives information about the electrophoretic Animals Laboratory of the Çapa Faculty of Medicine protein structures of venoms from a number of of İstanbul University. Th e 25 male rats were divided venomous snakes, including M. xanthina. However, into 5 groups (n = 5 animals per group). Th ese there are no histological studies available on the groups were used as the control (groups 1 and 2) damage caused by M. xanthina venom on tissues. Th e and assay (groups 3, 4, and 5). No application was

518 H. TOPYILDIZ, S. HAYRETDAĞ

made to the fi rst control group (control 1). Since the groups at 1, 3, and 6 h showed no signs of change. venom was diluted in physiological saline solution, In contrast, however, superfi cial dermis oedema and 10 μL of physiological saline solution (0.9% NaCl) collagen degeneration was observed on the dermis was injected into the right gastrocnemius muscle layer from all application groups (Figures 2-4). Th e of the second control group (control 2). Aft er the eff ect was 100% on the skins of the 1-h application intramuscular venom injection, the rats from the group and 80% on the skins of the 3-h and 6-h fi rst, second, and third assay groups were dissected 1, application groups (Table). Rare infl ammatory cells 3, and 6 h later, respectively. on the dermis and large haemorrhagic areas on the hypodermis appeared on all samples (Figures 2-3). Histopathological examinations Also evident in the same areas were infl ammatory Th e rats were anaesthetised with ether and dissected cell infi ltration and infl ammation, which had an through a cervical dislocation to take samples of intense mixed character, especially around the veins their skin, liver, and skeletal muscle tissues 1, 3, and (Figure 5). 6 h aft er the venom injection. Aft er being fi xed in Liver Bouin solution and following routine histological procedures, the tissues were embedded in paraffi n Th e sections taken from control group rat liver blocks. Sections 5 μm thick were taken from these tissues had a normal appearance (Figures 6a and blocks and stained with haematoxylin and eosin 6b). Congestion and hepatocellular degeneration (McManus and Moury, 1964). Stained preparations were detected within liver sinusoidal cells in all of were examined under an Olympus CX21 light the groups that were subjected to venom. Th is eff ect microscope; images were taken using an Olympus applied to 100% of the application groups (Table) BX51 light microscope and Olympus Analysis LS (Figures 7-9). soft ware. Muscle Th e muscular tissues from the control groups were Results of normal appearance (Figures 10a and 10b). In the samples of rat muscle tissues taken 1 h aft er the venom Skin application, haemorrhage, myonecrosis, and mixed Th e skin samples taken from the control 1 and character cell infi ltration were generally detected control 2 groups conformed to set norms (Figure in the perimysium and endomysium (Figures 11a 1). Th e epidermis layers from the experimental and 11b). Th e muscular tissues taken 3 h aft er the

100 μm 100 μm Figure 1. Skin section of rats in control group: epidermis (E), Figure 2. Rare infl ammatory cells (➡), oedema and collagen dermis (D). Stained with haemotoxylin and eosin degeneration (▲), fat necrosis (asterisk), and local (H&E). haemorrhage (➞) in the skin section of 1-h application group. H&E.

519 Histopathological eff ects of Montivipera xanthina venom on rats

100 μm 100 μm Figure 3. Rare infl ammatory cells (➡), oedema and collagen Figure 4. Rare infl ammatory cells (➡), oedema and collagen degeneration (▲), and local haemorrhage (➞) in the degeneration (▲), fat necrosis (asterisk), and local skin section of 3-h application group. H&E. haemorrhage (➞) in the skin section of 6-h application group. H&E.

Table. Incidence of observed lesions in the skin, liver, and muscle of male rats administered venom.

Time of venom application (h) Lesions 0136 Skin Haemorrhage 0 5 5 4 Oedema 0 5 5 5 Infl ammatory cell 0 5 5 5 Fat necrosis 0 5 5 5 Collagen degeneration 0 5 5 5 Liver Haemorrhage 0 5 5 5 Hepatocellular degeneration 0 5 5 5 Muscle Haemorrhage 0555 Mixed character infl ammatory cell inf.0555 Myonecrosis 0555 Formation of inclusions in myofi brils0555

Numbers indicate the number of animals observed with lesions in their tissues; n = 5. venom application showed that the formation of 12b). Th e muscular tissues taken aft er 6 h showed haemorrhage and myonecrosis increased, the mixed large areas of haemorrhage and myonecrosis, mixed character cell infi ltration in the perimysium and character strong cell infi ltration in the perimysium endomysium became more intense, and inclusions and endomysium, and formations of inclusions in in the myofi brils became evident (Figures 12a and the myofi brils (Table) (Figures 13a, 13b, 14).

520 H. TOPYILDIZ, S. HAYRETDAĞ

20 μm 100 μm Figure 5. Intense mixed character infl ammatory cell infi ltration Figure 7. Sinusoidal congestion (➡) and hepatocellular (➯) and infl ammation, especially around the capillary degeneration (white arrow) in the liver of 1-h vessel, in the hypodermis of 6-h application group. application group. H&E. H&E.

50 μm 100 μm Figure 8. Sinusoidal haemorrhage (➡) and hepatocellular Figure 6a. Liver section from rat in control group: central vein degeneration (white arrow) in the liver of 3-h (cv) and portal area (pa). H&E. application group. H&E.

50 μm 50 μm Figure 9. Sinusoidal haemorrhage (➡) and hepatocellular Figure 6b. Liver section from rat in control group: hepatocyte degeneration (white arrow) in the liver of 6-h (H) and central vein (cv). H&E. application group. H&E.

521 Histopathological eff ects of Montivipera xanthina venom on rats

50 μm 100 μm Figure 10a. Longitudinal section of muscle from rat in control Figure 10b. Transversal section of muscle from rat in control group: myocyte ({ ). H&E. group: perimysium (➡), myofi bril (➞). H&E.

50 μm 100 μm Figure 11a. Haemorrhage (➡), rare mixed character Figure 11b. Haemorrhage (➡) and myonecrosis (▲) in the infl ammatory cell infi ltration (➯), and myonecrosis transversal section of muscle of 1-h application (▲) in the longitudinal section of muscle of 1-h group. H&E. application group. H&E.

50 μm 100 μm Figure 12a. Mild mixed character infl ammatory cell infi ltration Figure 12b. Haemorrhage (➡), mixed character infl ammatory (➯) and myonecrosis (▲) in the transversal section cell infi ltration (➯), and myonecrosis (▲) in the of muscle of 3-h application group. H&E. transversal section of muscle of 3-h application group. H&E.

522 H. TOPYILDIZ, S. HAYRETDAĞ

100 μm 100 μm Figure 13a. Intense mixed character infl ammatory cell infi ltration Figure 13b. Haemorrhage (➡), intense mixed character (➯) and myonecrosis (▲) in the longitudinal section infl ammatory cell infi ltration (➯), and myonecrosis of muscle of 6-h application group. H&E. (▲) in the transversal section of muscle of 6-h application group. H&E.

20 μm Figure 14. Haemorrhage (➡), mixed character infl ammatory cell infi ltration (➯), and formations of inclusion in myofi brils (⇔) in the transversal section of muscle of 6-h application group. H&E.

Discussion was detected in their skin, liver, and skeletal muscle Although there are no specifi c data on the number tissues 1, 3, and 6 h aft er the injection intervals. of venomous snakebite cases in Turkey, it is Many pathological symptoms in skin tissue were estimated that a few people die from snakebite every detected in previous studies carried out using a variety year (Okur et al., 2001). Venomous snakebites are of diff erent snake venoms. In a study by Jimenez et especially common in rural areas. For this reason, al. (2008), Bothrops asper venom was injected into the clarifi cation of snake venom structure and its the skin around the ear area of rats. Th e authors eff ects on organisms is very important in terms of observed that oedema and haemorrhage were present administrating medication. In this study, rats were at 1- and 6-h intervals, respectively. In addition, 24 intramuscularly injected with M. xanthina venom h aft er the samples were taken from the application on an LD50 dose basis, and very serious damage group, collected data included loss of epidermis tissue,

523 Histopathological eff ects of Montivipera xanthina venom on rats

cell infi ltration, the presence of protein liquid, and cerastes of the family Viperidae, the results detected formation of scabs on the skin. Granule formation and were as follows: hyperaemia around the central vein, increasing epithelial cells were observed in the 72-h focal mononuclear infl ammation of white blood application group, and in the 7- and 14-day application cells, bleeding in sinusoids around the portal area, groups, the skin returned to its normal condition. In and hepatocyte necrosis tissue on the liver (Hanafy another study carried out with the same species, 3 h et al., 1999). Th e study found sinusoidal bleeding aft er intramuscular injection of venom, the epidermis and hepatocellular degeneration in the liver tissues and dermis layers slightly separated from each other of rats in all 3 application groups. Unlike previous with a low level of haemorrhage and cell infi ltration studies, however, it did not fi nd the formation of cell (Rucavado et al., 1998). A study using Naja nigricollis infi ltration, infl ammation, or necrosis. venom showed that the venom caused necrosis in Since venom is generally transferred by the snake the application area; infl ammation occurred around intramuscularly, the histopathological evaluations the veins and mixed character cell infi ltration had of muscular tissue are of special importance. Ali et taken place (Iddon et al., 1987). Contrary to the al. (2000) did some case studies on the venom of results found by Jimenez et al. (2008), in this study, Hydrophis cyanocinctus (sea snakes) and examined no damage to the epidermis was detected following the myotoxic eff ect of venom, which established the an intramuscular injection of M. xanthina venom. onset of myonecrosis and cell infi ltration. Th e eff ect of However, similar to previous studies, oedema on the Philodryas patagoniensis snake venom was analysed dermis and collagen degeneration were detected. Th is using albino rats. Th e results showed evidence of degeneration was widespread in tissues from the 1-h haemorrhage and myonecrosis in the gastrocnemius application group, whereas collagen degeneration was muscle in the 1-h application group and the addition generally local in the 3- and 6-h application groups. of intense infi ltration in the 3-h application group. All In all of the application groups, there was large-scale of these symptoms were more prominent in the 24-h haemorrhage in the hypodermis, fat necrosis, strong application group (Acosta et al., 2003). Similarly, Tu cell infi ltration, focal bleedings on the dermis, rare et al. (1969) observed that venom of Vipera russelli infl ammatory cells, and cell infl ammation around siamensis causes myonecrosis despite the fact that veins. During histopathological research, it was it does not cause haemorrhage in muscular tissues. established that the venom caused serious damage Th e venom of Vipera apis, on the other hand, causes to connective tissues in rats 1 h aft er injection and both haemorrhage and myonecrosis in muscular that this damage was partially restored in the 3- and tissue. It was established that the venoms of Bitis 6-h application groups. Elements in the M. xanthina gabonica and Bitis nasicornis cause haemorrhage, but venom structure cause bleeding by tearing vein walls not myonecrosis, in muscular tissue. Th e venom of as a result of haemolytic activity. Skin oedema, cell Bitis arietans brought about both haemorrhage and infi ltration, and infl ammatory reactions can be target myonecrosis in muscular tissue. Similar to previous symptoms for reducing the damaging eff ects of venom studies, there was haemorrhage seen in muscular on tissues. In addition, local necrotic areas can be tissues at the point of injection and evidence of mixed the result of the lytic activity of one or more venom character cell infi ltration. Th ere was myonecrosis in elements. all 3 application groups. When comparing application In analyses of the eff ects of Hydrophis cyanocinctus groups to each other, these symptoms were not very sea snake venom on liver tissue, Ali et al. (2000) intense in the 1-h application group; they were intense detected hepatocyte degeneration, infl ammation, in the 3-h application group and much more intense necrosis, fi brosis, regeneration, and cell infi ltration. in the 6-h application group. In the 3-h application Teibler et al. (1999) observed that Bothrops alternatus group, there were also inclusions within myofi brils, snake venom caused no damage to liver tissue. In a which were not detected in the 1-h application study on venom from Agkistrodon halys, a Pallas-type group; these became serious in the 6-h application , the fi brotic activity of rat livers was group. Some researchers have pointed out that the examined and hepatocellular damage was detected proteolytic enzymes available in snake venom bring (Chang et al., 2005). In another study that included about haemorrhage and necrosis in muscular tissues pathological symptoms from the venom of Cerastes (Flowers, 1963; Goucher and Flowers, 1964).

524 H. TOPYILDIZ, S. HAYRETDAĞ

As illustrated in this study, M. xanthina venom Acknowledgements is the cause of many pathological eff ects in diff erent Th is study was supported by the Çanakkale tissues. Due to the occurrence of these eff ects shortly Onsekiz Mart University Scientifi c Research Project aft er injection, it is very important to give snakebite Commission (2007/15 project). victims immediate medical attention. Hence, this study may enlighten practitioners/professionals as to Th is research is a part of an MSc thesis. Th e the eff ects of snake poisons and serve as a guide for authors wish to thank Professor Dr C. Varol Tok related medical treatment methods. for his assistance with the Montivipera xanthina description and the venom used in this study.

References

Acosta, O., Levia, L.C., Peichoto, M.E., Marunak, S., Teibler, P. and Hanafy, M.S., Rahmy, N.A. and Abd El Khalek, M.M. 1999. Th e Rey, L. 2003. Hemorrhagic activity of the Duvernoy’s gland dielectric properties of neutron irradiated snake venom and its secretion of the xenodontine colubrid Philodryas patagoniensis pathological impact. Phys. Med. Biol. 44: 2343-2366. from the north-east region of Argentina. Toxicon. 41: 1007- Iddon, D., Th eakston, R.D.G. and Ownby L. 1987. A study of the 1012. pathogenesis of local skin necrosis induced by Naja nigricollis Ali, S.A., Alam, J.M., Abbasi, A., Zaidia, Z.H., Stoeva, S. and venom using simple histological staining techniques. Toxicon. Voelter, W. 2000. Sea snake Hydrophis cyanocinctus venom. 25: 665-672. II. Histopathological changes, induced by a myotoxic Jimenez, N., Escalente, T., Gutierrez, J. M. and Rucavado, A. 2008. phospholipase A2 (PLA2-H1). Toxicon. 38: 687-705. Skin pathology induced by snake venom metalloproteinase: Arıkan, H., Kumlutaş, Y., Türkozan, O. and Baran, İ. 2003. acute damage revascularization and re-epithelization in a Electrophoretic patterns of some Viper venoms from Turkey. mouse ear model. J. Invest. Dermatol. 118: 1-8. Turk. J. Zool. 27: 239-242. Leonardi, A., Gubensek, F. and Krizaj, I. 2001. Purifi cation and Aznaurian, V.A. and Amiryan, S.V. 2006. Histopathological changes characterisation of two hemorrhagic metalloproteinases induced by the venom of the snake Vipera raddei (Armanian from the venom of the long-nosed viper Vipera ammodytes adder). Toxicon. 47: 141-143. ammodytes. Toxicon. 40: 55-62. Balija, M.L., Vrdoljak, A., Habjanec, L., Dojnovic, B., Halassy, B., McManus, J.F.A. and Mowry, R.W. 1964. Staining Methods: Vranesic, B. and Tomasic, J. 2005. Th e variability of Vipera Histologic and Histochemical. 1st ed. Hoeber International ammodytes ammodytes venom from Croatia - biochemical Reprint, London and Tokyo. properties and biological activity. Comp. Biochem. Physiol. Okur, M.İ., Yıldırım, A.M. and Köse, R. 2001. Türkiye’deki zehirli 140: 257-263. yılan ısırmaları ve tedavisi. T. Klin. Tıp Bilimleri 21: 528-532. Baran, İ. 2005. Türkiye Amfi bi ve Sürüngenleri. TÜBİTAK Popüler Rucavado, A., Nunez, J. and Gutierrez, J. M. 1998. Blister formation Bilim Kitapları, Ankara. and skin damage induced by BaP1, a haemorrhagic Baran, İ. and Atatür, M.K. 1998. Türkiye Herpetofaunası. T.C. Çevre metalloproteinase from the venom of the snake Bohtorpus Bakanlığı, Ankara. asper. Int. J. Exp. Path. 79: 245-254. Başoğlu, M. and Baran, İ. 1980. Türkiye Sürüngenleri Kısım II. Samel, M., Subbi, J. and Siigur, E. 2002. Biochemical characterisation Yılanlar. Ege Üniversitesi Fen. Fak. Kitaplar Serisi 81, İzmir. of fi brinogenolytic serine proteinases from Vipera lebetina snake venom. Toxicon. 40: 51-54. Bjarnason J.B. and Fox, J.E. 1989. Hemorrhagic toxins from snake venoms. J. Toxicol. Toxin Reviews 7: 121-209. Teibler, P., Acosta de Perez, O., Marunac, S., Ruiz, R., Koscinzuk, P., Sanchez Negrete, M. and Mussart de Coppo, N. 1999. Lesiones Chang, P., Hung, D.Y., Siebert, G.A., Bridle, K. and Roberts, M.S. localesy sistemicas inducidas por veneno de Bothrops alternatus 2005. Th erapeutic eff ects and possible mechanisms of a snake (vibora de la cruz) de Argentia. Acta. Toxicol. Argent. 7: 7-10. venom preparation in the fi brotic rat liver. Dig. Dis. Sci. 50: 745-752. Tu, A.T., Homma, M. and Hong, B.S. 1969. Hemorrhagic, myonecrotic, thrombotic and proteolytic activities of viper Flowers, H.H. 1963. Th e eff ects of X-irradiation on the biological venoms. Toxicon. 6: 175-178. activity of cottonmouth moccasin (Ancistrodon piscivorus) venom. Toxicon. 1: 131-136. Warrell, D.A. 1989. Snake venom in science and clinical medicine. 1. Russell’s viper: biology, venom and treatment of bites. Trans. R. Goucher, C.R. and Flowers, H.H. 1964. Th e chemical modifi cation Soc. Trop. Med. Hyg. 83: 732-740. of necrogenic and proteolytic activities of venom and the use of EDTA to produce Agkistrodon piscivorus, a venom toxoid. Toxicon. 2: 139-144.

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