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Abstract

Nutritional and therapeutic values of the edible gastropod (snail) are well known throughout the world. Earlier from this laboratory, a protein VB-P4 was identified from the edible snail (Vivaparous bengalensis) flesh extract having anti-osteoporosis and antiosteoarthritis activity on albino Wistar rats. The present study was done on the in vivo/in vitro toxicities of VB-P4 and heat treated VBP4 (HT-VB-P4) on experimental animals. The in vivo toxicities of VB-P4/HT-VB-P4 was done in male albino Swiss mice treated for 15 days and assessed through haematological (total count of RBC/WBC, Hb%), biochemical markers (SGOT, SGPT, LDH, CK-MB, urea, creatinine) and histological studies of organs (liver and kidney). The in vitro toxicities of VB-P4/HT-VB-P4 was done on isolated guinea-pig heart/auricle (for cardiotoxicity), isolated rat phrenic nerve diaphragm preparation (for neurotoxicity), haemorrhagic activity (in vivo cutaneous and gastric haemorrhage), haemolytic activity on RBC (mice, rat, guinea-pig and goat) and rat peritoneal mast cell degranulation activity. It was observed that 15 days sub-chronic treatment in male albino mice with VB-P4/HT-VB-P4 did not significantly changed the total count of RBC, WBC and haemoglobin. However, 15 days sub-chronic treatment in male albino mice with VB-P4 significantly increased the liver markers (SGOT, SGPT), myotoxicity markers (LDH, CK-MB) but not the renal markers (Urea, Creatinine). HT-VB-P4 did not significantly change the liver markers (SGOT, SGPT), myotoxicity markers (LDH, CK-MB) and the renal markers (Urea, Creatinine). Histological studies with VB-P4 showed significant changes in liver tissue (fatty infiltration, necrotic lesions, dilated central vein) as compared with the control group of mice. Histological studies with VB-P4 did not show any significant changes in kidney tissue. In vitro cardiotoxicity was shown by VB-P4 on isolated guinea-pig heart but not on isolated guines-pig auricle. VB-P4 significantly decreased the heart rate and amplitude of contraction leading to irreversible blockade of contraction. HT-VB-P4 did not showed cardiotoxicity on the above preparations. VB-P4/HT-VB-P4 did not show neurotoxicity on isoalated rat phrenic-nerve diaphragm preparation. VB-P4 induced cutaneous haemorrhagic spots, gastric lesions in male albino rats through in vivo studies, which was not found with HT-VB-P4 treatment. Histological studies of stomach treated with VB-P4 confirmed the presence of blood, gastric lesions and necrotic spots, which was absent in HT-VB-P4 treatment. In in vitro studies, VBP4 produced haemolysis of RBC (mice, rat, guinea-pig and goat) and degranulated rat peritoneal mast cells, which was absent in case of HT-VB-P4 treatment. The data from this study confirmed that intake of raw snail flesh protein may induce toxicity especially in liver, stomach and RBC. Boiling/cooking before consumption may overcome the toxicities and safe for human consumption.

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Keywords

Viviparous bengalensis; Edible snail; Snail protein; in vivo toxicity; in vitro toxicity

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Introduction

Viviparous bengalensis (VB), a fresh water edible snail, is a staple food for the socio-economically backward people of rural India as a cheap protein substitute source. In many countries (Philippines, Vietnam, Laos, Cambodia, China, Japan, northeast Indian states), people eat snail flesh in raw form as delicacy. In Indonesia, they are fried as satay, a dish known as sate kakul. In many countries, people use to boil snail in hot water before consumption. In India, snail cooked in mustard oil, used as an ointment over different types of sore and corn. In the north Bihar state of India, the flesh of Bellamia (Viviparous) bengalensis had been used as a traditional medicine against arthritis [1]. Snail flesh contained different types of protein, peptide, lipoproteins, oligosaccharides, enzymes of biological importance [2]. Gauri et al. purified and characterized a novel antibacterial peptide from Bellamia bengalensis which was found to be active against ampicillin and chloramphenicol resistance Staphylococcus epidermitis [3]. Sarkar et al. reported that Indian edible snail (Viviparous bengalensis) flesh extract possess antiosteoporosis and anti-osteoarthritis activity in experimental rats [4]. Recently, Sarkar et al. isolated a protein (VB-P4) from the snail (Viviparous bengalensis) flesh extract which was found to possess anti-osteoporosis and anti-osteoarthritis activity in albino rats [5,6]. VB-P4 had 562 amino acids, glycoprotein in nature and acted through pro/anti oxidants, serum cytokines and through T cell modulation. The present study was an effort to explore the toxic (both in vivo and in vitro) effect of fresh water edible snail flesh protein VB-P4 on experimental animals.

All the materials and chemicals used were of analytical grade, unless otherwise mentioned. The following chemicals were used in the present study: Acetic acid, Anaesthetic ether, disodium hydrogen phosphate, DPX, Eosin, Folin phenol reagent, Haematoxylene, NaCl, sodium dihydrogen phosphate, Toluidine blue, Tri sodium isocitrate (SRL, India), Bovine serum albumin (Sigma-Aldrich, USA), CK-MB Kit, Creatinine Kit, LDH Kit, SGOT Kit, SGPT Kit, Urea Kit (Labkit, India), Drabkins reagent (Cogent, India), Ethanol (Bengal Chemicals, India), Paraffin (Merck, Germany).

Swiss albino male mice (20±2 g), male albino Wistar rats (150±10 g) and male guinea pig (200±20 g) were procured from enlisted supplier of Calcutta University and housed in standard polypropylene cages at controlled environment (24 °C±2 °C, 12 hour light-dark cycle, relative humidity 60%±5%). The animals were provided with dry pellet diet, green vegetables, gram and water ad libitum. The experiments were conducted according to the departmental animal ethics committee for the purpose of control and supervision of experiments on animals (animal ethical reference no. PHY/CU/ IAEC/20/2008). All animal experiments were approved by the animal ethics committee, Department of Physiology, University of Calcutta and were in accordance with the guideline of the committee for the purpose of control and supervision of experiments on animal (CPCSEA), Government of India. For in vitro RBC hemolysis, goat blood was collected from slaughter’s house.

Viviparous bengalensis live sample collection and purification was done as described earlier [4]. The snails (Viviparous bengalensis) were identified by the Zoological Survey of India. Snail flesh was dissected out and a 10% of the flesh homogenate was prepared in PBS (0.01 M, pH 7.2) using a glass homogenizer fitted with a Teflon piston (Remi Motors Ltd; India, model no. RQ-127A). The homogenate was centrifuged (8000 rpm x 30 min at 4 °C) and the supernatant was termed as Viviparous bengalensis extract (VBE) expressed in terms of protein content. Crude VBE was subjected to ion exchange chromatography (IEC) using DEAE cellulose column with phosphate buffer. Fractions were collected using graded NaCl molarity and all the fraction proteins were estimated after Lowry et al. [7]. Peak 4 (VBP4) was further analyzed through HPLC using C18 column (4.6×250 mm), methanol:water solvent system (70:30) with a flow rate 1 ml/ min and monitored at 280 nm.

VB-P4 was kept at 80±2 °C for 20±2 min, cooled and centrifuged at 3000 rpm for 15 min. The supernatant was collected for protein estimation after Lowry et al. [7]. It was provisionally designated as HT-VB-P4 (heat treated Viviparous bengalensis protein 4).

In vivo toxicity studiesGrouping of animals and treatment: Male Swiss albino mice (20±2 g, n=6) were taken and divided into 3 groups- Gr.1: Control, Gr.2: VB-P4 treated (40 μg/20 gm /i.p/15 days) and Gr.3: HT-VB-P4 treated (40 μg/20 gm /i.p/15 days). On 16th day, blood was collected from hepatic portal vein (for haematological studies) and serum was separated for biochemical studies. Tissues (liver, kidney) were collected for histological studies.

Hematological and biochemical parameters: For hematological studies, blood was collected in heparinised vial and total count of WBC, RBC and hemoglobin percentage was done after Mukherjee et al. [8]. Serum was prepared from blood and SGOT, SGPT, Urea, Creatinine, LDH, CK-MB were analyzed with biochemical kit (Labkit, India) using UV spectrophotometer (Analab, model no UV-180).

Histological studies: Liver, kidney and stomach tissues were collected, fixed in 10% buffered formalin for 24 hrs. The tissues were then dehydrated in graded (50-100%) ethanol followed by clearing in Xylene. Paraffin (56-58 °C) embedding was done at 58±1 °C for 4 hrs, followed by paraffin block preparation. Paraffin section (5 μ) was cut using a rotary microtome (Weswox Optic, India). Paraffin sections were deparaffinised with xylene, stained with hematoxylineosin, followed by mounting in DPX with a cover slip. Histological characteristics of the tissues were observed with a bright field microscope (Motic, Germany) and photographs were captured with Motic software (Motic Images Plus 2.0 software).

In vivo rat cutaneous haemorrhage: This was performed according to the method of Kondo et al. [9]. VB-P4 (10 μg/i.d/20 gm mice/24 hrs) and HT-VB-P4 (10 μg/i.d/20 gm mice/24 hrs) was injected on the shaved dorsal flank on the Swiss albino male mice (n=4). Same amount of phosphate buffer (pH 7.4) was injected incontrol animal. The mice were sacrificed after 24 hour, skin was opened and observed for hemorrhagic lesion. Diameter of haemorrhagic spot was measured with a caliper and photograph was captured with a digital camera (Panasonic Lumix). Minimum haemorrhagic dose (MHD) was the dose of VB-P4 that produced a haemorrhagic spot of 10 mm diameter.

In vivo rat gastric haemorrhage/lesion: Male albino Wistar rats (150±10 g, n=4) were deprived of food for 72 hour (water was provided ad libitum) before experiment. The animals were lightly anaesthetized with anesthetic ether. A small slit was made in the abdomen and stomach pylorus ligation was done after Shay et al. [10]. The animals were orally fed with VB-P4/ HT-VB-P4 (40 μg/per oral/100 g rat) and after 4 hr, all the rats were sacrificed and the hemorrhagic spot, gastric lesion was photographed with a digital camera (Panasonic Lumix). Stomach tissue samples were fixed in formal-buffer for histopathological studies following the method as stated earlier.

Effect on isolated guinea pig heart and auricle: Isolated guinea pig heart was prepared after Langendorff [11] and perfused with oxygenated (95% O₂ + 5% CO2) Tyrode’s solution (NaCl 137 mM, KCl 2.7 mM, CaCl₂·2H₂O 2.6 mM, MgCl₂·6H₂O 1.27 mM, NaHCO₃ 11.9 mM, NaH₂PO4 0.4 mM and glucose 5.5 mM) containing double dextrose (2 g l-1), temperature was maintained at 37±1 °C. The preparation was treated with VB-P4/HT-VB-P4 (40 μg/bolus) and contraction was recorded on a rotating smoked drum using a heart lever.Isolated guinea pig auricle was prepared after Burn and suspended in oxygenated Tyrode’s solution (29 ±1 °C) in a 4 ml glass bath [12]. The preparation was treated with VB-P4 and HT-VB-P4 (10 μg/4 ml bath volume) and spontaneous contraction was recorded on a smoked drum by a lightly sprung lever.

Effect on isolated nerve-muscle preparation: Isolated rat phrenic nerve diaphragm (RPND) was prepared after Bulbring and suspended in a 6 ml glass bath containing oxygenated (95% O2 + 5% CO2) Tyrode’s solution at 29±1 °C [13]. The preparations were stimulated with a square wave electronic stimulator of 8 V, 0.5 ms duration, 0.2 Hz (Grass, USA, Model No. SD9). RPND preparations were treated with VB-P4 and HT-VB-P4 (20 μg/5 ml bath volume) and contraction was recorded with Brodie’s lever on a smoked kymograph paper.

In vitro RBC haemolysis: Hemolytic activity of VB-P4/ HTVB-P4 was done according to Rothschild [14]. Blood (mice, rat, guinea-pig and goat) was collected in 3.8% citrate buffer, repeatedly washed with 0.9% saline and a 2% suspension of RBC (in 0.9% saline) was prepared. 1 ml RBC suspension was treated with VB-P4/ HT-VB-P4 (10-50 μg/ml RBC suspension), incubated at 37 °C for 30 min. Volume was made with chilled 0.9% saline and centrifuged at 3000 rpm for 15 min. The extent of hemolysis was estimated in the supernatant fluid (against saline treated control) at optical density of 540 nm. Distilled water was used as standard (100% lysis). Haemolytic dose 50 (HD50) was calculated graphically with the mice RBC haemolysis % vs. doses of VB-P4.

In vitro rat peritoneal mast cell degranulationPeritoneal membrane was collected in 0.9% saline from a freshly killed male albino Wistar rat. The Peritoneal membrane was cut in small pieces and placed in different watch glasses in 0.9% saline. Group 1: 0.5 ml 0.9% saline (control), Group 2: 0.5 ml 0.9% saline and VB-P4 (10-80 μg/ml) and Group 3: 0.5 ml 0.9% saline and HT-VB-P4 (10-80 μg/ml). The watch glasses were incubated at 37 °C for 30 min. After incubation, the peritoneum from each group was placed on clear glass slide in perfectly stretched condition and was fixed over flame. The slide was then treated with ethanol:chloroform:acetic acid (6:3:1) mixture for 15 min. After downgrading treatment with ethanol:water, the slides were stained with 0.1% aqueous toluidine blue. The excess stain was washed with distilled water and upgrading treatment with ethanol:water up to 100% ethanol. The slides were washed twice with xylene and mounted in DPX. The mast cell count was done under the light microscope and expressed in terms of % degranulation against control preparation [15]. Mast cell degralulation dose 50 (MCD50) of VB-P4 was calculated against saline treated control preparation.

Data were expressed as mean±SEM (n=6). Statistical analysis was done using one way analysis of variance (ANOVA). P< 0.05 was considered to be statistically significant.