SYNANCEIA VERRUCOSA) ENVENOMATION in BALB/C MICE

BIOCHEMICAL AND HISTOLOGICAL OBSERVATIONS OF LUNG INJURY AFTER STONEFISH (SYNANCEIA VERRUCOSA) ENVENOMATION IN BALB/c MICE

Mohammad Wahsha1,*, Tariq Al-Najjar2, Haya Al-Tarawneh3, Maroof Khalaf2, Amer Saad4

1Marine Science Station, The University of Jordan, Aqaba Branch, Jordan 2Department of Marine Biology, The University of Jordan, Aqaba Branch, Jordan 3Control Health Division, Aqaba Special Economic Zone Authority, Aqaba, Jordan 4Armed Forces Hospital, Southern Region, Khamis Mushait, Saudi Arabia

ABSTRACT hidden under the sand or between coral make them difficult to detect and avoid [7]. Stonefish are usually This study aimed to evaluate the toxicity of sluggish and will not attack a human being unless the stonefish venom isolated from genus Synanceia ver- dorsal spines are trampled upon [5]. As a result of rucosa, collected during winter 2015 and spring stonefish envenomation injuries symptoms appeared 2016 along the Jordanian coastline of Gulf of Aqaba, to be; respiratory difficulty due to pulmonary oe- Red sea. Stonefish venom was isolated from the dema, hypotension, bradycardia, arrhythmia, cardio- venom glands of the dorsal spines. Venom was in- vascular collapse, muscle weakness, paralysis con- tramuscular injected in experimental animal BALB vulsion and death at severe injuries [8, 5, 6]. Respir- c/mice in order to evaluate the adverse effect of the atory system composed of; the conducting tract (tra- venom on mice lung tissues by studying histological chea, bronchi and bronchioles) and the respiratory changes and the biochemical effect on the Alanine zone (respiratory bronchioles, alveolar ducts, and al- Aminotransferase (ALT) enzymatic activity. Results veoli), and gas exchanging is the main function of showed that the median lethal dose of the extracted lungs [9]. Respiratory bronchioles are joined to thin- venom was 0.107mg venom/kg mice body weight. walled gas exchange areas lined by alveolar epithe- Histological results showed pathological develop- lium, alveolar duct, alveolar sacs (clusters of alve- ments among the selected tissues. Lunge showed de- oli), and alveoli. Alveoli are separated by interalve- formation in overall structure, severe inflammation, olar septa, connective tissue sheets that contain a ca- hemorrhage and cellular degeneration. Moreover, pillary bed, which covered by alveolar epithelium significant increasing in the enzymatic activities of and supported with pore of kohn [10, 9]. Pores of Alanine transaminase (ALT) was found as a result of Kohn provide collateral ventilation, which maintains cellular injuries caused by the stonefish venom. The an equal pressure across neighboring alveoli and pre- observed pathological changes reflect the possibili- vents atelectasis; they allow the passage of immune ties of failure in the respiratory system functions cells, fluid, and infectious agents. The alveolus is the with time as a result to stonefish venom. functional and structural component of the respiratory zone [9]. As respiratory system is vital organ and being affected with toxins, the current study KEYWORDS: aimed to evaluate the toxicity of stonefish venom on Synanceia verrucosa, Gulf of Aqaba, Stonefish. lungs, using histological and biochemical ap- proaches based on [5, 8, 9, 11].

INTRODUCTION MATERIALS AND METHODS A total of 14 fish species belonging to the family Scorpaeindae occurred along the Jordanian coast Chemicals. All chemicals used in this study [1]. Stonefish belongs to the family scorpaenidae were analytical grade and purchased from Sigma and to genus Synanceia, found in tropical waters (Pa- Chemical Company, USA unless otherwise indi- cific Ocean, Indian Ocean and Red Sea); it is one of cated. the most venomous fish in the world [2, 3, 4]. They have defense system composed of 16 spines (13 dor- Fish sample. A total of 10 fish samples of sal) supported with venom glands and (3 anal) [5, 6]. Synanceia verrucosa were collected by SCUBA div- Stonefish inhabit the shallow water they founded ing from northern sites of the Gulf of Aqaba and kept near the bottom, among the corals and rocks or alive in oxygenated seawater aquarium at the aqua- within sand [3, 4]. They have strong ability to cam- culture unit in Marine Science Station (MSS). Fish ouflage which in addition to their habits for being

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species were identified according to Khalaf and Disi from all groups and treated with formalin for fixa- [3]. tion. Samples were dehydrated and embedded according to the procedures described by Al-Haj [16]. Experimental animal. 60 Male BALB/c mice Tissue sections (7µm) were stained using hematox- 6-7 week's old, average body weight 25gm were ylin and eosin stain (H&E), and analyzed microscop- used. Mice were obtained from animal house at Yar- ically based on Conti et al. [10], Al-Haj [17] and mouk University and were maintained on standard Treuting and Dintzis [18]. laboratory diet and tab water during the experiment procedure. Statistical analysis. Statistical analysis was based on ANOVA and is presented as means ± S.D. Isolation of fish crude venom. Venom was Statistical significance was considered at p-value of isolated by inserting rubber caps of the test tubes into 0.05 or less. The data were analyzed statistically us- the spine of the fish; reaching the suitable distance in ing Sigma Stat statistical software version 3.5. the spine will extrude the high viscous venom. A volume of 0.3-0.4 ml of venom was dissolved in 1ml phosphate buffer saline, to get milky diluted venom. RESULTS The extracted soluble crude venom was immediately o stored in a dark container at -20 C for further histo- The concentration of the crude stonefish venom biochemical analysis. The concentration of the ex- was calculated using the standard curve and found tracted venom was determined according to Lowry 4.466µg/ml. The LD50 value of the extracted stone- et al. [12]. fish venom was evaluated using BALB/c mice under laboratory conditions. The approximate LD50 of ex- LD50 determination. A modified Fawell's up tracted toxin was 0.107 mg toxin/kg mouse body and down method [13] was adopted to evaluate the weight. intramuscular injection (i.m) LD50 value of the ex- After the i.m injection, mice exhibited abnor- tracted venom using BALB/c mice under laboratory mal behavior and it was distinguished by losing of conditions, in order to establish an adequate dose of their energetic activity and difficulty breathing, sub- venom to investigate its toxic effects. Thirty times sequently sufficient to cause detectable paralysis and (V/V) of stock solution of crude stone fish venom clearly increased after the second hours. was diluted and concentrations of 0.01, 0.05, 0.1, 0.150, 0.155 and 0.200 V/V were initially used. This Stonefish venom effect on Alanine Transam- process was continued until the approximate LD50 inase (ALT) activity. Control mice group (C) exhib- has been determined. The experiment was set into ited normal levels of ALT activity and it was 0.8u/l. triplicate groups of healthy mice (n=10). Figure. 1 illustrate a statistically significant increase in ALT activity in the lungs of the toxin treated Mice bioassay. According the method de- groups T1, T2 and T3 respectively when compared to scribed by Wahsha et al. [14], 60 male BALB/c mice the control group (P<0.05). ALT activity increased 6-7 weeks-old (average body weight 25g) were used. by 18, 45 and 21 folds among T1, T2 and T3 respec- They were divided into two main groups: The first tively as a result of stonefish venom. group (C) was the control group; mice were envenomed by intramuscular injection (i.m) with phos- Lung histology results. Control groups phate buffer saline without venom (15 mice). The showed normal regular distribution of its compo- second group (T) was the toxin treated groups (45 nents as alveolar capillaries, alveoli and alveolar mice); mice were envenomed by intramuscular in- walls (Figure 2). Histological changes appeared in jection (i.m) with 0.107 mg toxin/kg mouse body the lungs of toxin treated (LD50) mice compared to weight (according to LD50 Value) and were subdi- the control mice. The lung tissues of the treated vided into three group: 15 mice were sacrificed after groups showed deformation in the overall organiza- 1hr (T1), 2 hours (T2) and 3 hours (T3). tion, areas of hemorrhage, inflammation and alveo- Lungs were removed immediately after scarifi- lar wall thickening (Figure 3). Venom treated group cation and divided into two portions. The first por- reveals less number and distribution of the pore of tion was perfused with normal saline containing hep- kohn. arin, and homogenized with phosphate buffer saline (pH 7.2). The perfused samples were kept in dark plastic bottles and stored at -20ºC for estimating the DISCUSSION effect of stonefish venom on Alanine aminotransferase (ALT) enzyme activity in the lungs. ALT assay Kuntz and Kuntz [19] and Ozer et al. [11] re- was carried out according to the procedure described ported that each organ possesses a typical quantita- by Vozarova et al. [15]. Regarding the histological tive and qualitative distribution of enzymes (enzy- observations, small pieces of lungs were collected matic pattern); and this was in agreements with our ALT results.

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Our histological results showed that the lungs Santos et al. [21] investigated the effect of Scor- in the toxin treated groups showed deformation in paena plumieri venom on the lungs; they found that the overall organization, areas of hemorrhage, in- BALB/c mice developed neutrophilic infiltrates, ar- flammation and alveolar wall thickening when com- eas of lung hemorrhage and alveolar macrophage ac- pared to lungs of control group. Treated lungs lost tivation within 24 hours after injection with S. plum- significant numbers of pore of kohn. Suarez et al. [9] ieri venom. These histopathological changes were reported that these pores acts as holes in the alveolar associated with an early increase in Bronchoalveolar septa and provide collateral ventilation, equalizing lavage fluid protein and early induction of cytokines, pressure between neighboring alveoli. They are also chemokines and matrix metaloproteinases, followed responsible for the passage of immune cells and flu- by a later increase in Bronchoalveolar lavage fluid ids within the tissue. In this context, and in agree- neutrophils. ment with previous study by Gwee et al. [5], one of the most symptoms of a stonefish sting is severe dif- ficulties in breathing as a result of losing of the lungs CONCLUSIONS function. This could be explained as a result of the disturbance occurred in the overall tissue organiza- The lethal dose (0.107mg/kg mice body tion. Moreover, our results showed that the respira- weight) of stonefish venom is capable of stimulating tory failure is a common feature in experimental en- sever damaging reaction, noted by the significant el- venomation by stonefish venom and this is in agree- evation of the cell injury biomarker (ALT). A nota- ments with the findings of Gwee et al. [5] and Khoo ble damage of lung tissue was occurred as a result of [6]. Furthermore, our results are also in agreement stonefish envenomation, and it's proportional to the with Gwee et al. [5] where the author noted that the dose of venom and elapsed time. Further researches injection of stonefish venom into various species of are necessary to evaluate the effect of stonefish animals might accompanied by irregular and de- venom in other organs and it is important also to pressed respiration, leading to the respiration failure study the distribution of stonefish along the coastline when lethal doses are used. of the Gulf of Aqaba in order to developing national Moreover, our histological results confirmed antivenom. that most piscine venoms are chemically similar, in agreement with Church and Hodgson [20]. Boletini-

45,0 40,0 35,0 30,0 25,0

20,0 ALT (µ/l) ALT 15,0 10,0 5,0 0,0 C T1 T2 T3

Lungs of miceALT group(u/l)

FIGURE 1 Trend of changes in ALT activity for mice lungs tissues. C: Control group, T1: Toxin group after 1hr, T2: Toxin group after 2hrs and T3: Toxin group after 3hrs. Presented data are mean values (units per liter) for each group mice ± SD. n= 7 mice.

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FIGURE 2 Histological structure of the lungs of Balb/c mice. Control groups showing normal alveolar capillaries, alveoli and alveolar walls and well organized structure are shown (H&E, 40X). 1: Alveolus, 2: Alveolar macrophage, 3: Type I Alveolar cell, 4: Type II Alveolar cell, 5: Alveol sac, 6: Pore of kohn, 7: Alveolar septa.

FIGURE 3 The histopathological changes in lungs of venom-treated (LD50) mice compared to the normal mice. Toxin treated lungs showed deformation in the overall organization, areas of hemorrhage, inflammation and alveolar wall thickening and less pore of kohn (H&E, 40X). C: Control group, T1: Toxin group at 1hr, T2: Toxin group at 2hrs, T3: Toxin group at 3hrs. 1: Alveoli, 2: epithelial wall, 3: Pore of kohn. *: Area of hemorrhage.

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