Biomedical and Pharmacological Potential of Tetrodotoxin-Producing Bacteria Isolated from Marine Pufferfish Arothron Hispidus (Muller, 1841)
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The Journal of Venomous Animals and Toxins including Tropical Diseases ISSN 1678-9199 | 2010 | volume 16 | issue 3 | pages 421-431 Biomedical and pharmacological potential of tetrodotoxin-producing ER P bacteria isolated from marine pufferfishArothron hispidus (Muller, 1841) A P Bragadeeswaran S (1), Therasa D (1), Prabhu K (1), Kathiresan K (1) RIGINAL O (1) Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai, Tamil Nadu, India. Abstract: Specimens of the pufferfish Arothron hispidus collected at Parangipettai, on the southeast coast of India, were subjected to bacterial isolation and identification. Three species were identified, namely Bacillus sp., Kytococcus sedentarius and Cellulomonas fimi. Partially-purified microbial filtrates exhibited hemolytic activity on chicken and human erythrocytes of O, B and AB blood groups, with maximum activity of 32 HU. The microbial filtrates also presented ATPase, Mg2+-ATPase, Na+K+-ATPase and AchE enzymatic activities of positive neuromodulation in Kytococcus sedentarius with 1300, 300.1, 1549.98 and 140.55%, in Cellulomonas fimi with 620, 300, 10 and 128.42%, and in Bacillus species with 40, 200, 849.98 and 158.69%, respectively. Toxicity symptoms were observed when the bacterial filtrate was intraperitoneally injected into mice. The bacterial filtrate caused adverse effects on viability of the mouse muscle cell line (L929) and leukemia cell line (P388). Maximum level of inhibition was observed on the growth of L929 cell line. Bacillus lentimorbus inhibited the cell line from 84.03 to 94.43% whereas Bacillus species inhibited the growth in a range between 77.25 and 86.16% at the lowest dilution. Key words: Arothron hispidus, tetrodotoxin, bioassay, ATPase and AchE enzymatic activities, anticancer activity. INTRODUCTION ability to inflate rapidly, filling their extremely elastic stomach with water (or air when outside Tetrodotoxin (TTX) is the best known marine the water) until they are almost spherical in shape. toxin due to its frequent involvement in fatal The bacteria associated with some pufferfish food poisoning, unique chemical structure and produce powerful neurotoxin in their internal specific action of blocking sodium channels of organs making them an unpleasant, possibly excitable membranes (1, 2). The toxin derives its lethal, meal for any predator (9). name from the pufferfish family (Tetraodontidae) and occurs widely in the terrestrial and marine MATERIALS AND METHODS animal kingdom (3-6). Tetrodotoxin is a powerful neurotoxin that can cause death in 60% of humans Specimen Collection (7). A human has to ingest only a few milligrams The puffer fishArothron hispidus (Muller, 1841) to produce a fatal reaction to the toxin (8). In (Vertebrata: Actinopterygii: Tetraodontiformes: recent years, tetrodotoxin has been utilized as a Tetraodontidae) was collected from the pharmacological probe. Annankoil fish landings at Parangipettai (Lat. As a defense mechanism, pufferfish have the 11° 29’ N; Long. 79° 46’ E) southeast coast of Bragadeeswaran S, et al. Biomedical and pharmacological potential of tetrodotoxin-producing bacteria isolated from pufferfish India, and immediately brought fresh to the Isolation and Partial Purification of TTX from laboratory (1 km). Skin, intestine and liver Bacterial Cultures tissues were aseptically excised for isolation of The pure isolate was inoculated with a sterile microbes. loop inside a capped 1,000-mL flask containing 500 mL of sterile Zobell broth medium for Isolation and Identification of Bacteria culturing at 25°C in an incubator for ten days in ZoBell marine agar 2216 (Himedia, Mumbai) darkness without aeration (4); subsequently, it was used for bacterial isolation. The agar plates was subjected to TTX detection. The isolation and were prepared in sterilized dishes with 15 g of purification of TTX from bacterial cultures were agar dissolved in 1.0 L of ZoBell 2216 medium, basically the same procedures as those used for adjusted to a pH of 7.2. One gram of pufferfish pufferfish tissues, briefly described by Yu (10), in skin, liver and intestine each was homogenized which each broth culture was centrifuged at 3,000 and suspended in 9 mL of sterile sea water. rpm for 15 minutes to remove the precipitated The samples were serially diluted for isolation bacterial cells. of bacterial strains by spreading 0.1 mL of the The supernatant was evaporated under diluted samples on agar plates and the result reduced pressure to a volume of water-washed was maintained at 28°C for 5 to 14 days to activated charcoal under agitation and filtered permit bacterial growth. Eventually, mixtures of through a funnel. The charcoal on the funnel was colonies were formed on each plate, after which thoroughly washed with distilled water and the they were purified by the streak plate method TTX adsorbed was eluted three times with three on another new ZoBell 2216 agar plate until volumes of 1% acetic acid in 20% aqueous ethanol. discrete colonies were obtained. Each discrete The eluent was evaporated and lyophilized. The colony was further sub-cultured several times to lyophilized toxin extract was dissolved in 10 mL ensure that it was a pure culture before bacterial of 0.03 M acetic acid and subjected to gel filtration identification using the Microbial Identification through a column (2 x 50 cm) of Bio-Gel® P-2 System (MIS version n. 4.5, Microbial (Bio-Rad Laboratories, USA) equilibrated with Identification Inc., USA), which identified 0.03 M acetic acid. Toxic fractions were combined the whole cell fatty acid profiles between the and concentrated at reduced pressure to a final bacterial samples and standards. volume of 10 mL which was subjected to further toxicity bioassay. Identification by the Microbial Identification System Hemolysis Study The microbial identification system relies The microbial crude extracts were assayed on on qualitative and quantitative analysis of chicken and human (A, B, AB and O) erythrocytes the culture strains identified by the fatty acid as described by Prasad and Venkateshvaran (11). composition of the organism (MIS version 3.8). Samples of chicken blood were obtained from a Moreover, a gas chromatograph (Agilent GC nearby slaughterhouse in Parangipettai, while 6890N®, Agilent Technologies, USA) equipped the human blood was obtained from Sheena with a flame ionization detector and a 2-meter Clinic, Andheri (W), Mumbai, using 2.7% HP Ultra 2 capillary column (HP 6890N®, ethylenediaminetetraacetic acid (EDTA) solution Agilent Technologies, USA) (inner diameter, as an anticoagulant at 5% of the blood volume, 0.2 mm; film thickness, 0.33 mm) coated with and brought to the laboratory. The blood was 5% phenyl methyl silaxane were used. The rate centrifuged thrice at 5,000 rpm for five minutes. of hydrogen carrier gas flow was maintained A 1% erythrocyte suspension was prepared for at 30 mL/minute. Fatty acid methyl esterases hemolysis study. (FAMEs) were identified by the Microbial The hemolytic test was performed in 96-well ‘v’ Identification (MIDI) calibration standard bottom microtiter plates. Serial two-fold dilutions software. The pure culture strains from the of the crude toxin/fractions were made in 100 μL third quadrant streaking (harvesting) culture of normal saline. Then, 100 μL of 1% erythrocyte were transferred into a tube with a Teflon-lined was added to all the wells. The positive and screw cap. The microbe was identified based on negative controls were maintained by adding 100 the fatty acid profile. μL of distilled water and 100 μL of normal saline, J Venom Anim Toxins incl Trop Dis | 2010 | volume 16 | issue 3 422 Bragadeeswaran S, et al. Biomedical and pharmacological potential of tetrodotoxin-producing bacteria isolated from pufferfish respectively, to the 1% red blood cell suspension. ATPase assay The plate was gently shaken and allowed to The ATPase assay for inorganic phosphate was stand for three hours at room temperature. performed according to Lowry and Lopez (13). Presentation of uniform red-color suspension in For total ATPase reaction mixture, 0.8 mL of the wells was considered to constitute positive imidazole buffer (0.135 mm) with 100 mM NaCl, hemolysis and a button formation in the bottom 20 mM KCl and 5 mM MgCl, were taken in each of the wells constituted an absence of hemolysis. test tube and 0.1 mL of enzyme (a quantity that The reciprocal of the highest dilution of the crude depends on the protein mg/h of enzyme source) toxin showing the hemolytic pattern (hemolytic was added and stirred. To this mixture, 0.1 mL of unit) was divided by the protein content to obtain toxin at each of four concentrations (250 μg, 500 the specific hemolytic unit. μg, 750 μg, and 1,000 μg) was added immediately by using micropipettes. Mouse Bioassay for Lethality For the Mg2+-ATPase reaction mixture, 0.07 All animal bioassays were carried out by mL of obtain (1 mM) was added as inhibitor for following the statement of the Institutional Na+-K+-ATPase in addition to the above mixture. Ethics Committee (regulation number 160/1999/ Triple distilled water (0.1 mL) was added to the CPCSEA/11.01.2008) of the Rajah Muthiah total ATPase reaction mixture and 0.03 mL of Medical College, Annamalai University. The triple distilled water was added to Mg2+-ATPase bioassay for lethality was done by using clinically mixture to bring the reaction mixture to a total healthy male albino mice weighting 20 ± 2g that volume of 1.05 mL. were maintained in a healthy condition in the The reaction was started by adding 50 μL of laboratory. Mice in triplicate sets were challenged ATP substrate (4.5mM) in each tube. All the intraperitoneally with 0.25, 0.50, 0.75 and 1.0 tubes were gently shaken and incubated at 37°C mL of the crude toxin, dissolved at 5 mg/mL and for 30 minutes in a water bath. By adding 0.5 mL 1.0 mL each of the 11 fractions.