Journal of the Egyptian Society of Parasitology, Vol.43, No.2, August 2013 J. Egypt. Soc. Parasitol., 43(2), 2013: 447 - 456 HISTOPATHOLOGICAL CHANGES IN LIVER OF MICE AFTER EXPER- IMENTAL ENVENOMATION WITH ANDROCTONUS AMOREUXI SCOR- PION VENOM By HAMDY A. FETAIH1, NAHLA M. SHOUKRY2, BELAL A. SOLIMAN2, MAHMOUD E. MOHALLAL3 and HOWAYDA .S. KHALED2 Department of Pathology1, Faculty of Veterinary Medicine, and Department of Zoology3, Faculty of Science, Suez Canal University1,3, and Department of Zoology2, Faculty of Science, Suez University, Suez Abstract A total of 78 adult male Albino mice were divided into thirteen groups (6 mice in each). One served as a control group and the other twelve groups were venom treated groups. The mice of treated groups were injected with 0.1 ml saline solu- tion in which a particular amount of venom. The first 6 groups were sub- cutaneously injected with 1/2 LD50 (0.05 g/g body weight), while the other 6 groups were injected with 1/4 LD 50 (0.025 g/g body weight) by the same route. The from each group were anesthetized with ethyl ether and sacrificed at different time intervals (3, 6, 9, 12 hrs, 4 & 7days post toxin administration). The microscopic examination of liver tissue obtained from envenomed animals showed variable histopathological changes being severely increased with the time interval of envenoming. The most obvious changes in the liver were acute cellular swelling, hydropic degeneration, congestion of central veins and portal blood ves- sels. Besides, extramedullary hematopoiesis and invaginations in nuclei of hepatic cells, with formation of intranuclear cytoplasmic inclusions were observed. Key words: Egypt, Androctonus amoreuxi, venom, histopathology, liver. Introduction to avoid or reduce their dangers are common venomous (Shoukry and Morsy, 2011). animals in temperate desert and tropi- In Egypt, there are about 13 poisonous cal habitats of the world. They can be species of scorpions some of them are found in the sea, up mountains, in both deadly poisonous (Abdel-Rahman et desert and the tropics, in caves, just al, 2010). Seven genera such as leivrus, above anywhere accept perhaps the Androctonus (family: ), Scor- Arctic and Antarctic (Fet et al, 2000). piomaurus (family: Scorpionidae) and Scorpions are a world health problem Nebo uierichonticus (family: Diplocen- in tropical and subtropical regions trida) were collected from St. Catherine (Abbass et al, 2009). Great attention and Wadi Feiran regions (Ibrahim and must be directed towards such animals Abdel-Rahman, 2011).

447 Androctonus amoreuxi scorpion is circulatory, degenerative and necrotic moderately abundant in many places in changes in the liver. South Sinai and highly distributed in By literatures review, none was pub- Upper Egypt. It is also common on the lished on the hepatic histopathological deserts of North Africa and South West effects of Androctonus amoreuxi scor- Asia. It usually called fat tail scorpion, pion venom. The present study aimed which produce one of the most potent at the evaluation of mice hepatic and important venom of Buthidae. A. changes after Androctonus amoreuxi amoreuxi scorpion considered among envenomation. medically important species in the world. It belongs to family Buthidae, Materials and Methods genus Androctanus and species amo- The crude venom was obtained by reuxi (Fet, 1997). The clinical manifes- the electric stimulation of the scorpion tations of scorpion envenomation vary telson every week by using electric among different species. The severity current according to the method de- of envenomation depends on the sting scribed by (Deodoras and Vad, 1962). site as well as species, size and degree Clean laboratory bred adult male of agitation of the scorpion and symp- Albino mice weighting 20+3.9 gm. toms only develop after 30 minutes and were purchased from Theodor Bilharz sometimes only after 4 to 12 hrs, in- Research Institute, Giza, Egypt. They creasing in severity over the following were transferred to wire-bottomed cag- 24 hrs. Most of scorpion sting result in es at the house of Zoology De- no more than to local pain, lasting to partment, Faculty of Science, Suez Ca- about 4 hours (De Roodt et al, 2001). nal University. The animals were kept The distribution of the venom in the at an optimal temperature and fed on a body of the envenomed victims (Dos- special rodent diet supplied by Medical gupta et al, 1991; Revelo et al, 1996) is Professions for the Veterinary Products variable. In general, the target organs and Fodders Additions Company of the venom scorpions of Family (MUVCO). The mice were given fresh Buthidae are heart, brain, blood, lungs, water through a glass bottle with a ca- liver and kidney (Bhatt et al., 1988). pillary dropper fixed to the cage wall in Scorpion venom is composed of vary- an available a position. The water was ing concentration of neurotoxin, cardi- changed and the cages were cleaned otoxin, nephrotoxin, hemolytic toxin every day. The mice were weighted and hepatotoxic (Nair et al., 1973; Ma- just before the beginning of each ex- rie and Ibrahim, 1976; Rochat et al, periment. 1979). Histopathological studies car- Determination of the LD50 of the ried out by Correa et al. (1997), Bessa- Crude Venom: The LD50 of the crude lem et al. (2003), Naser et al. (1992) venom was calculated according to and Patel et al. (1992) on another scor- Meier and Theakston (1986). The ly- pion species, proved the occurrence of ophilized crude venom was reconstitut- ed with physiological saline solution

448 (1mg venom/5ml physiological saline with hematoxylin and eosin (Drury and solution; 0.85% NaCl) then diluted into Wallington, 1980). the following concentration: 0.12, 0.16, Results 0.2, 0.24, 0.28, 0.32, 0.36, and 0.4 g/g The histopathological changes in- body weight. 0.1 ml from each concen- duced in liver were recorded in mice tration was subcutaneously (SC) inject- envenomed with 1/4 LD and 1/2 LD ed into adult male Albino mouse 50 50 doses of the crude venom. The effects (20+3g in weight) respectively. After were studied after 3, 6, 9, 12 hrs, 4 & 7 injection, the survival time (T) of each days post-envenomation, and compared injected mouse (D) was recorded. Re- with control ones to delineate the se- gression line was plotted by using the quence of events caused by the venom values of D/T versus D. The constant a at different time intervals of envenom- (Slope) and b (LD ) of the regression 50 ing. line was calculated. Light microscopic examination of the Experimental groups: a- Control liver tissues obtained from envenomed group: Six mice were subcutaneously animals showed variable histopatholo- injected with 0.1 ml saline solution to gical changes being severely increased be used as control animals. b- Enveno- with the time interval of envenoming med group: Seventy two mice were (Tab. 1). divided into twelve groups (6 mice in each). Mice of all subgroups were in- Discussion jected with 0.1 ml saline solution in Gross pathological examination re- which a particular amount of scorpion vealed that the body organs of the en- venom, 6 group were subcutaneously venomed animals varied according to injected with 1/2 LD50 (0.05 g/g the time interval between venom injec- body weight), while the other 6 groups tion and observation. The observed were injected with 1/4 LD 50 (0.025 hepatic congestion after 3- 6 hrs from envenoming with different doses of g/g body weight) by the same route. Androctonus amoreuxi venom was Animals from each group were anes- previously observed by Correa et al. thetized with ethyl ether and sacrificed (1997) due to injection of Tityus serru- at different time intervals (3, 6, 9, 12 latus scorpion venom. hrs, then after 4 and 7 days, following the venom administration. The present study clarified that injec- tion of A. amoreuxi scorpion venom Histopathological examination: After induced several serious histopathologi- scarification, liver was collected from cal alternations in the hepatic tissue of all animals fixed in neutral buffered envenomed mice. The severity and de- formalin 10% for 3 days at room tem- gree of extent of these alterations were perature, dehydrated in different grades dose and time dependent. of ethanol, cleared in zylol, embedded in paraffin wax, and then sectioned at 4microns. The sections were stained

449 Fig.1: Histopathological changes of mice liver. (A): Control liver section showed cords of hepatocytes surrounding central veins (CV), and hepatic sinusoids (HS). (B): 6 hrs post-injection (PI) with 1\4 LD50 of venom showing congestion of blood vessels (CB). (C): After 4 days (PI) with 1\4 LD50 showed hydropic degeneration (HD), some nuclei of hepatocytes display karyolysis (KL) and karyorhexis (KH). (D): After 9 hrs (PI) with 1\2 LD50 showed extramedullary hematopoiesis (arrows) and dilatation of hepatic sinusoids (DS). (E): After 4 days (PI) with 1\2 LD50 showed dilated blood vessels (DB) with fibrinoid degeneration (FD) and deposition of faint basophilic ho- mogenous material in the portal area (F): After 7 days (PI) with (PI) with 1\4 LD50 showing hepatocyte- and kary- omegally (arrows) (H&E, X200)

450 C I

Fig, 2: (A): liver section of mouse after 12 hrs of subcutaneous injection of Androctonos amorenxi venom (1/2 LD50) showed convoluted nuclei (CN) of some hepatocytes and loss of chromatin in others i.e. necrotic cells (NC) besides some hepatocytes display karyorhexis (KR) and karyolysis (KH). (B): After 9 hours of subcutaneous injection of Androctonos amorenxi venom (1/2 LD50) showing necrotic cells (NC), nuclear invagination (NI) and apoptic bodies (arrows). (C): After 12 hrs of subcutaneous injection of A. amorenxi venom (1/2 LD50) showed convoluted nuclei of some hepatocytes with intranuclear cytoplasmic inclusios (CI), besides loss of chromatin in others i.e. necrotic cells. (H&E, 400 X)

451 Alterations included variable degrees enlargement of hepatocytes may be due of cellular swelling, cytoplasmic chan- to hypertrophy of heptocellular smooth ges, cellular necrosis and cellular dam- endoplasmic reticulum. The dilatation age accompanied with loss of common of cisternae of the smooth endoplasmic architecture the hepatic parenchyma at reticulum was reported (Anderson and different stages of the envenoming. Ownby 1997). Dilatation of the endo- Cytoplasmic granularity and vacuoliza- plasmic reticulum could be attributed tion of hepatocytes with any doses after to the accumulation of positive sodium 3-6 hrs agreed with (Omran and Abdel- ions within the cells as well as entrance Rahman, 1992). They revealed cyto- of extra-cellular water to the cell due plasmic granulation and vacuolization disturbance of the cell membrane per- suggesting hydropic degeneration. Cy- meability induced by the venom toplasmic vacuolization was in hepato- (Krimm et al, 1999). Also, the present cytes of animals envenomed with T. study showed obvious increase in size discrepans venom (D'Suze et al, 2004). of some hepatocytes and their nuclei, Cytoplasmic granularity and vacuole- megalocytosis, (cytomegaly and kary- zation were accompanied by swelling omegaly). Megalocytosis characterized of the hepatocytes after injection of ¼ by large number of swollen hepato- and ½ LD50 doses. Similar results cytes with abundant granular eosino- were reported (Correa et al, 1997) in philic cytoplasm and occasional large rats injected with fractions from the nuclei (Kwon et al, 2007). venom of Tityus serrulatus scorpion. The karyolysis and karyorrhexis of the Bessalem et al. (2003) illustrated the nuclei of envenomed mice could repre- degenerative changes and enlargement sent an advanced stage of nuclear of most of the hepatic cells in mice changes, which may be attributed to envenomed with activation of endonucleases enzymes hector scorpion. Enlargement or swell- located within the nuclei of the injured ing of the hepatocytes could be the cells (Williams et al, 2000). main cause of constriction of the hepat- One of the obvious alterations of ic sinusoids and the disturbance of the scorpion envenomation in the present architecture of the hepatic parenchyma. study was presence of extramedullary Similar observations were recorded in haematopoiesis in the liver. Demasi et liver tissues of animals envenomed al. (2003) stated that the preferential with Androctonus crassiceuda (Jacob- uptake of zinc by edema fluid proteins son and Jacobs, 1992) as well as Lei- lead to increase in zinc metabolism and urus quinquestriatus (Omran and Ab- activated macrophages. Activated mac- del-Rahman, 1992) scorpions. Cellular rophages release cytokines which in swelling might be due to the action of turn stimulates of pro-inflammatory venom phospholipase, which causes peptides which increase vascular per- disturbance in cell membrane per- meability. Extramedullary haemato- meability with consequent influx of Na poiesis in the liver was one anatomical and water (Segelke et al, 1998). The marker of hypoxemia the function of

452 activated macrophage with cytokines cytosis, nuclear vesicular and nucleolar regulation. The present study showed prominence, cytoplasmic intra-nuclear that the liver of envenomed mice suf- inclusions and cytoplasmic bile accu- fered from many abnormal mitotic fig- mulations (Jessen et al, 2003). In this ures. There is either clumping or dis- study, the fibrinoid degeneration and persal of chromatin, and these appear hyperplasia of endothelial cell of portal to be mitotic onset or late metaphase. blood vessels was detected. Fibrinoid The interpretation of mitotic disturb- degeneration tissues accumulate depos- ance (Maxie, 2007) after pyrolizidine its of an acidophilic homogeneous ma- alkaloids which inhibit DNA synthesis terial that resembles the fibrin when and mitosis in the hepatocytes, but stained. Fibrinoid material does usually some are able to replicate their DNA contain fibrin and tends to be eosino- without under-going mitosis, resulting philic (staining with acidic dye eosin). in greatly enlarged hepatocytes with However, in some cases the fibrinoid large convoluted nuclei. Some enlarged deposits may contain the significant nuclei have cytoplasmic invaginations amounts of the nuclear debris, includ- that can become entrapped as intra- ing acidic DNA and may be hematox- nuclear inclusions. However, many yphilic (staining blue with the basic hepatocytes in an affected liver do not dye hematoxylin) and these can be ex- become megalocytic. Those completely plain the presence of deposition of ho- inhibited do not replicate DNA at all, mologous basophilic materials in the whereas those that are more resistant portal area, around bile ducts and in can replicate more normally and give sub-endothelium of the blood vessels. rise to nodular population of smaller, Fibrin indicates that the nearby blood more normal hepatocytes. Inhibited vessels have become highly permeable hepatocytes and megalocytes are long- and often destroyed (Kumar et al, lived but eventually many undergo 2005). apoptosis. The present study showed El Nasr et al. (1992) reported that the presence of intranuclear inclusions main toxic effect of B. quinquestriatus spherical, retractile eosinophilic struc- scorpion venom was primarily on the tures resulted in chronic injured liver liver blood vessels of as manifested by under effect of hypoxia or intoxi- dilatation of branches of the hepatic cations. Interpreted of such phenomena arteries and the portal veins together that hepatocytes during autolysis can with the occurrence of signs of hemor- imbibe plasma that forms similar round rhage. Many other authors ensured the but the less eosinophilic, and non- presence of hemorrhage in case of en- membrane-bound cytoplasmic inclu- venoming by different types of scorpi- sions. The hepatoxicity develops more ons (Mohamed et al, 1978; Patel et al, slowly than in response to toxicants 1992; Zare et al, 1994). Light micros- that induce membrane per-oxidation, as copy of liver sections did not show many hepatocytes with DNA damage hemorrhage. undergo apoptosis, and some megalo-

453 References rpiones, Buthidae) captured in Argen- Abbas, N, Laegros, C, Brigitte, C, tina. J. Nat. Toxins 10, 2:99-109. Belghazi, M, Hamon, A, Boughs, PE, Demasi, M, Cleland, LG, Cook, RJ, et al, 2009: Full characterization of 3 Caughey, G, James, M, 2003: Effects toxins from Androctonus amoreuxi of hypoxia on monocyte inflammatory scorpion venom. Toxicon 54: 460-70. mediator production: Dissociation bet- Abdel-Rahman, MA, Omran, MA, ween changes in cyclooxygenase-2 ex- Abdel-Nabi, I, Nassier, O, Schemer- pression and eicosanoid synthesis. J. horn, B, 2010: Neurotoxic and cyto- Biol. Chem. 278, 40: 38607-16. toxic effects of venom from different Deodoras, PJ, Vad, NE, 1962: The populations of Egyptian Scorpiomaur milking of scorpions. Toxicon 1:41-8. us palmatus. Toxicon 55, 2/3: 298-306 Dosgupta, S, Gomes, A, Lahiri, SC, Anderson, SG, Ownby, CL, 1997: 1991: Detection and distribution of Systemic hemorrhage induced by prot- scorpion (Heterometrus bengalensis) einae from Crotalus adamanteus (Eas- venom in rabbit tissues by enzyme- tern diamondback rattlesnake) venom. linked immuno-sorbent assay (ELISA). Toxicon 35, 8:1301-13 Indian .I. Exp. Biol. 29, 7:69-2. Bessalem, S, Hammoudi-Triki, D, Drury, M, Wallington, A, 1980: Car- Laraba, DF, 2003: Effect of immuno- lton’s Histological Techniques. Oxford therapy on metabolic and histopatho- University Press, Oxford. logical modifications after experiment- D'Suze, G, Moncada, S, Gonzalez, C, tal scorpion envenomation. Bull.Soc. Sevcik, C, Aguilar, V, et al, 2004: Re- Pathol. Exot. 96, 2:110-4. lationship between plasmatic levels of Bhatt, VH, Mehta, NS, Panchal, CM, various cytokines, tumor necrosis fact- 1988: Serum biochemical profile dur- or, enzymes, glucose and venom con- ing scorpion venom (Leiurus quinque- centration following Tityus scorpion straitus) in toxication in weaning sting. Toxicon 41, 3:367-75. Charles foster rats. Cent. Afric. J. Med. El Nasr, MS, Abdel Raliman, M, Sh- 34, 8:173-80. oukry, N, Ahmed, M, et al, 1992: Eff- Correa, MM, Sampaio, SV, Lopes, ect of scorpion envenomation on diffe- RA, Mancuso, LC, Cunha, OAB, et rent organs of albino mice. J. Egypt. al, 1997: Biochemical and histopatho- Soc. Parasitol. 22, 3:833-8. logical alternations induced in rats by Fet, V, 1997: Notes on the (Titlus serrulatus) scorpion venom and of some old world scorpions. J. Ara- its major neurotoxin Tityustoxin-I. chnol. 25:245-50. Toxicon 35, 7: 1053-67. Fet, V, Sisson, WD, Lowe, G, Braun- De Roodt, AR, Gimeno, E, Portians- Walder, ME, 2000: Catalogue of Sco- ky, E, Varni, L, Dolab, J, et al, 2001: rpions of the World (1758-1998). The A study on the experimental enveno- Entomological Society, New York. mation in mice with the venom of Ibrahim MM, Abdel-Rahman MA, Tityus trivitattus Kraepelin 1898 (Sco- 2011: Natural infestation of Pimeliaph-

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455 dose 3 hrs. 6 hrs. 9 hrs. 12 hrs. 4 days 7 days 1\4 - Congestion of central - Congestion of - Congestion of - Moderate - Liver tissue exhibited - LD50 veins and portal blood central veins (F. 1, central veins. congestion of central diffuse hydropic Megalocytos vessels. B). - Hydropic vein degeneration (F. 1, c), is with - Acute cellular swelling - Acute cellular degeneration of -Megalocytosis of with occasional pyknotic karyomegaly of hepatocytes with swelling of liver cells. hepatocytes nuclei. (F. 1, F). granular cytoplasm. hepatocytes with - Pyknotic nuclei - Karyomegaly of - congestion. - Clumping of nuclear granular of some nuclei of numerous - necrobiotic chromatin in most cytoplasm. hepatocytes cells. changes in hepatocytes - Karyomegaly - Necrobiotic changes hepatocytes. -Karyomegaly with with marked indicated by clumping marked hyperchromacia of hyperchromacia of of nuclear chromatin. nuclei in numerous cells. few cells. - 1\2 - Congestion of central - Same as in 3 hrs - Extramedullary - Mild congestion of - Fibrinoid degeneration - Same as in LD50 veins and portal blood group. hematopieosis. (F. central veins. & deposition of 4 days group vessels. 1, D) - Hepatocytomegally homogenous basophilic & detection - Dissociation of - Degeneration of with obliteration of materials in portal area, of apoptotic hepatocytes. liver cells with sinusoids. around bile ducts & in bodies & - Cytomegaly of some granularity of - Hyper-chromatic subendothelium of blood some cells, mostly subcapsular. cytoplasm. nuclei vessels (F.1- E) necrotic - Karyomegaly with - Nuclear - Nuclear - Marked swelling in cells. marked hyperchromacia of invaginations invaginations with endothe-lial of blood nuclei in numerous cells. with intranuclear intranuclear vessels. - Atypical mitotic figures cytoplasmic cytoplasmic - Some nuclei showed inclusions (F. inclusions (F. 2, A nuclear invaginations & 2,B) &C). others showed convoluted contour. - Extramedullary hematopoiesis.

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