Indian Journal of Experimental Biology Vol. 47, December 2010, pp 1225-1232 Biological activity of sea anemone proteins: I. Toxicity and histopathology Vinoth S Ravindran 1*, L Kannan 2 & K Venkateshvaran 3† 1, 2 Centre of Advanced Study in Marine Biology, Annamalai University, Portonovo 608 502, India 3Aquatic Biotoxinology Laboratory, Central Institute of Fisheries Education, Versova, Mumbai 400 054, India Received 1 May 2008: revised 12 July 2010 The crude as well as partially purified protein fractions from anemone species viz. Heteractis magnifica, Stichodactyla haddoni and Paracodylactis sinensis , collected from the Gulf of Mannar, south east coast of India were found to be toxic at different levels to mice. The mice showed behavioral changes such as loss of balance, opaque eyes, tonic convulsions, paralysis, micturiction, flexing of muscles, prodding (insensitive to stimulii), foaming from mouth and exophthalmia. The toxic proteins upon envenomation produced several chronic and lethal histopathological changes like formation of pycnotic nuclii and glial nodules in the brain; heamolysis, thrombosis and myocardial haemorrhage in the heart; granulomatous lesions, and damage to the hepatic cells in the liver and haemorrhage throughout the kidney parenchyma and shrinkage of glomerular tufts in the kidney. The toxins proved to be neurotoxic, cardiotoxic, nephrotoxic and hepatotoxic by their action on internal organ systems. The toxins were also thermostable till 60 oC and had considerable shelf life. Keywords: Mammalian toxicity, Sea anemone protein, Thermostable Sea anemones are ocean dwelling sedentary organism the male albino mice 7. The report also exhibits the belonging to Phylum Cnidaria, Class Anthozoa. toxin to have caused haemorrhage in the brain, They produce toxic polypeptides and proteins. haemolysis in the heart and kidney and severe damage More than 40 toxic peptides have been isolated to the hepatocytes in the liver. Inspite of all these from different sea anemones 1,2 . The understanding explorations from these organisms, the sea anemones of envenomation requires recognition of the gross from the Indian waters have been poorly studied for manifestations seen in organs removed from the body their toxicity. The present study has been therefore, and an appreciation of the underlying microscopic undertaken to obtain information on the toxicity of changes 3 in correlation with clinical signs and three sea anemone species viz. Heteractis magnifica symptoms 4,5 . Hence histopathological investigations (Quoy and Gaimard, 1833) , Stichodactyla haddoni are inevitable to reveal the effect of the toxin on (Saville-Kent, 1893) and Paracodylactis sinensis specific organs. Carlgren 1934, collected from the Gulf of Mannar, Studies regarding the histopathalogy of the southeast coast of India. animals, envenomed by sea anemone toxins are very meager. Limited reports are there on the Materials and Methods histopathalogical effects caused by the sea anemone Extraction of crude —The anemones were collected toxins. Fatal damage to the liver cells of a young man, from the field and 500 g fresh weight of body tissue who was stung by an anemone, has been reported 6. was macerated and extracted with 500 ml of Incidentally, it was the only known case of death methanol, which was evaporated to dryness in a 8 caused by sea anemone envenomation. The toxin of Rotary Flash Evaporator at 37°C and the extracts the sea anemone Anthopleura middori caused severe were stored at -20°C until further analysis. damage to the brain, heart, kidney and liver cells of Partial purification of the crude extract —Partial —————— purification of the crude extract (5mg/ml) was *Correspondent author (present address): carried out using DEAE Cellulose Anion Exchange Suganthi Devadason Marine Research Institute, Chromatography 9. Ten fractions were collected in a 44 Beach Rd. Tuticorin 628 001, India step-wise gradient with 0.1-1M NaCl in phosphate Telephone: (0) 9655638373; 0461-2336487/88 Fax: 0461-2325692 buffer saline (PBS). The collected fractions were E-mail: [email protected] stored at -20°C for further use. 1226 INDIAN J EXP BIOL, DECEMBER 2010 Protein estimation —Protein estimation 10 was done, Results using bovine serum albumin (BSA) as the standard. Crude extract—The amount of crude extract The absorbance was read spectrophotometrically at obtained from 500 g fresh weights in each case 280 nm. was 9.73 g in H. magnifica, 7.84 g in S. haddoni and Mice bioassay for lethality—Kausauli strain male 5.37 g in P. sinensis. albino mice of 20±2 g body weight procured from Protein content—Protein content in the crude M/s. Haffkine Biopharma, Mumbai, were maintained extract was highest in H. magnifica followed by in a healthy condition in the animal house, following S. haddoni and P. sinensis . Amount of protein in the the codal formalities of Central Institute of Fisheries purified fractions also followed a similar trend as Education, Mumbai, in accordance with the norms of the crude (Table 1). Animal Welfare Ethics. Mice bioassay for lethality—Crude extract of the anemones containing 1.25, 2.50, 3.75 and 5.0 mg of The crude toxin dissolved @ 5 mg/ml in PBS protein when injected ip to mice showed symptoms of was injected ip to the test mice in doses containing toxicity but the levels at which they were lethal varied 1.25, 2.50, 3.75 and 5.0 mg of toxin. Also, 1.0 ml of from species to species. The dose at which the crude each fraction (protein content mentioned in Table 2) and fractionated proteins were toxic have been was injected ip to different test mice. Triplicate sets tabulated (Table 2). were maintained for each dose. The injected mice Behavioral changes in mice—The changes in were kept under observation in mice rearing cages. behaviour of the envenomed were: lying on belly with The time of injection and the time of death were widespread forelimbs, running around the cage in an recorded using a seconds-timer stop-watch, besides exited manner, escape reaction, prolonged palpitation, recording the behavioral changes before death. closed eyes, grooming, shivering of fore limbs, loss of Mice that died upon envenomation were autopsied balance, opaque eyes, squeaking, tonic convulsions, to observe gross anatomical changes such as gasping for breath, arching of body backwards, hemorrhage, blood clots, septicemia, dark or pale paralysis, micturiction, flexing of muscles, prodding discoloration of internal organs, etc., if any. (insensitive to stimulii), diarrohea, lethargy, dragging Stability of toxin—Bioassays were conducted to of hind limbs, rolling of tail, foaming from mouth and ascertain the stability of the crude anemone proteins exophthalmia. However, interestingly, when certain from H. magnifica , S. haddoni and P. sinensis, as fractions such as fraction 5 from H. magnifica , affected by (i) heating at different temperatures fraction 6 from S. haddoni and fraction 5 from (50, 60, 80 and 100°C) (ii) at different levels of pH P. sinensis were injected, the mice became very brisk (3.0-8.0) and (iii) storing at –20°C for more than one and active after envenomation and all these fractions year. In the first case, samples were heated to the were not lethal. above said temperature on a water bath for 5 min 9. Stability test —Lethal activity of the crude proteins Each of these samples was immediately tested for the was not affected on storage for 14 months at -20 °C. toxicity by ip injection with the lethal dose to male The crude extracts were found to be thermolabile. They albino mice as described above. In the second case, were stable up to 60 °C after which they lost their pH of the samples was adjusted using 0.1 N HCl Table 1—Protein content (µg/ml) of crude and partially purified or 0.1 N NaOH to pH levels 3.0, 4.0, 5.0, 6.0, 7.0 and fractions of sea anemones 8.0. From the adjusted toxin solutions, lethal dose [Values are mean±SE of triplicate sets] was immediately injected ip to mice to ascertain Fraction no. H. magnifica S. haddoni P. sinensis the toxicity. Crude 981.1±0.32 820.4±0.37 605.3±0.31 Histopathology—Brain, heart, liver and kidney 1 70.5±0.11 30.3±0.24 110.6±0.33 were dissected out from mice that died upon 2 160.7±0.25 30.6±0.19 10.3±0.14 envenomation while ascertaining the toxicity of the 3 70.3±0.18 140.8±0.27 50.4±0.18 4 80.6±0.17 10.2±0.13 10.3±0.11 anemone extracts. The dissected organs were fixed in 5 20.3±0.13 20.5±0.11 20.2±0.16 10% formalin for a minimum period of 24 h and 6 30.7±0.21 60.4±0.23 10.2±0.13 processed for histopathological observations 5. 7 10.9±0.17 10.1±0.09 10.6±0.06 Prepared sections were examined and photographed 8 10.7±0.08 10.2±0.10 10.7±0.08 under a microscope (Labomed, CX II). 9 20.4±0.15 10.3±0.06 10.4±0.11 10 10.2±0.10 10.4±0.11 10.2±0.11 RAVINDRAN et al .: BIOLOGICAL ACTIVITY OF SEA ANEMONES : TOXICITY & HISTOPATHOLOGY 1227 Table 2—Toxicity of crude extracts of the anemones injected ip at different doses to male albino mice Species Quantity injected Amount of protein (µg/kg) Death time (sec) (ml) In the injected sample ( µg/ml) Toxic dose (µg/kg) (mean±SE) H. magnifica 0.25 245.27 - - - 0.50 490.55 - - - 0.75 735.83 - - - 1.0 980.1 49,005 68.3±2.60 S. haddoni 0.25 205.1 - - - 0.50 410.2 - - - 0.75 615.3 30,765 135±2.64 1.0 820.4 - - - P.