Introduction
The consequences of jellyfish blooms have concerned scientists and environmental managers due to the increasing incidence of blooms in the late of 1990s and their significant threat to fishery and swimmers in Korean Seas including the Yellow Sea, East China Sea, and East Sea [1]. Recently, four species of scyphozoan jellyfish (Nemopilema nomurai, Cyanea nozakii, Aurellia aurita and Rhopilema esculentum) widely distributed in coastal areas of Korea. Among them, N. nomurai (order Rhizostomeae) is one of the largest jellyfish in the world, and is capable of growing to a bell diameter of 2 m and a weight of 200 kg [2]. R. esculentum (order Rhizostomeae), C. nozakii (order Semaeostomae) and A. aurita (order Semaeostomae), they are reaching up to 70, 50, and 15 cm in bell diameter, respectively [3, 4]. The venoms of each jellyfish are stored and discharged by nematocysts and contain bioactive proteins that are differed in activity and composition [5]. The symptoms of these jellyfish envenomation can produce skin erythema, swelling, burning and small vesicles, and occasionally severe dermonecrotic, cardio-depressant effects, which are sometimes fatal for some patients[4,6]. At present, over than 2,000 cases of jellyfish envenomation and 13 fatal cases reported in coastal areas of Korea, China and Japan since 1983 [1].
Most researches are presently focused on the relationship between the scyphozoan jellyfish blooms and the ocean ecosystem [7], but have not documented the four species of scyphozoan jellyfish venoms for their comparative toxicity. Recently, we reported cytotoxic and hemolytic activities in vitro of the venom from N. nomurai jellyfish that these activities were dose-dependent both in mammalian cell culture lines and several animal erythrocytes preparations [8, 9]. The use of cytotoxicity and hemolytic activity assays enable the venom to be studied over varying concentrations in the same assay which is not possible in whole animal studies. This allowed us to rapidly compare a large number of venoms at different concentrations to find potentially minor differences in potency. Moreover, these in vitro functional assays will be able to be correlated with their in vivo potencies and thus aid in the identification of the as yet unknown lethal factors in the venoms. The aim of this study was to give a reliable and comparable description of the venoms of four scyphozoan jellyfish by investigating their cytotoxicity, hemolytic potency, brine shrimp toxicity and protein components.
Materials and Methods
Chemicals and reagents
Dulbecco's Modified Eagle's Medium (DMEM), penicillin, streptomycin sulfate, trypsin, dimethyl sulphoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Alsever's solution and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). All other reagents used were of the purest grade available.
Jellyfish collection and preparation
Four different of jellyfish specimens were captured from various geographical locations around the coast of Korea Strait as follows, N. nomurai from the coasts of Tongyoung; C. nozakii from the coasts of Mokpo; A. aurita from Jeju island; R. esculentum from the Yellow sea near Kanghwado. The tentacles dissected from the jellyfish were stored in ice and transferred immediately to our laboratory for further preparation. Nematocysts were isolated from the dissected tentacles as described by Bloom et al. [10] with a slight modification. In brief, tentacles were gently swirled with the addition of distilled water, then stood still for 1?2 hr to remove debris and sea water. After decanting the supernatant, tentacles settled down at the bottom were mixed with 2 × (v/v) distilled water and shaken vigorously for 3 min. The detached nematocysts were separated by filtering tentacle preparation through 4 layers of medical gauze. This was repeated for two more times with additional distilled water to harvest nematocysts from the tentacles. The filtrates were centrifuged (700 × g) at 4℃ for 20 min and the pellets (nematocysts) were lyophilized and stored ?20℃ until use.
Venom extraction and preparation
Venom was extracted from the freeze-dried nematocysts using the technique described by Carrette and Seymour [11] with a minor modification. In brief, venom was extracted from 50 mg of nematocyst using glass beads (approximately 8,000 beads; 0.5 mm in diameter) and 1mL of ice-cold (4℃) phosphate buffered saline (PBS, pH 7.4). These samples were shaken in a mini bead mill at 3000 rpm for 30-s intervals for five times with intermittent cooling on ice. The venom extracts were then transferred to a new eppendorf tube and centrifuged (22,000 × g) at 4℃ for 30 min. This supernatant was used the present study. Protein concentration of the venom was determined by the method of Bradford technique [12] (Bio-Rad, C.A. USA) and the venom was used based on its protein concentration.
Cell culture and cytoxicity assay
C2C12 (mouse skeletal myoblast) and H9C2 (rat heart myoblast) cells were used for assessing the cytotoxic activity of the jellyfish venoms. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin at 37℃ with 5% CO2. The cells were seeded in 24-well plates at a density of 104 cells/well and cultured for 24 hr. Non-adherent cells were removed by gentle washing with fresh culture medium and venoms were treated at the various concentrations. After incubation (24 hr), cytotoxicity was assessed by measuring mitochondrial dehydrogenase activity, using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Briefly, 100 μl of MTT solution (5 mg/ml) was added to each well and incubated for another 3 hr at 37℃. After removing the supernatant, the formazan crystal generated was dissolved by adding 250 μl/well of dimethyl sulfoxide (DMSO) and the absorbance was detected at 540 nm using a spectrophotometric microplate reader (BioTek Instruments, Inc., Winooski, USA).
Hemolysis assay
Hemolytic activity of the venom was tested using the erythrocytes of dog, human and rat. In brief, freshly collected blood samples were immediately mixed with anticoagulant, Alsever's solution (pH 7.4) to prevent blood coagulation. To obtain a pure suspension of erythrocytes, 1 ml of whole blood was then made up to 20 ml in PBS, and centrifuged at 1,500 × g for 5 min at 4℃. The supernatant and buffy coats were then removed by gentle aspiration, and the above process was repeated two more times. Erythrocytes were finally resuspended in PBS to make 1% solution for hemolysis assay. For this, indicated concentration of each jellyfish venoms (1 mg/ml) were added to the suspension of red blood cells obtained from dog, human and rat. The venom-erythrocyte mixtures were incubated at 37℃ for 1 hr in water bath and then centrifuged at 1,500 × g for 5 min at 4℃. The supernatants were transferred to 96-well microplates and the absorbance at 415 nm was determined by using a spectrophotometric microplate reader (BioTek Instruments, Inc., Winooski, USA) to measure the extent of red blood cell lysis. Positive control (100% hemolysis) and negative control (0% hemolysis) were also determined by incubating erythrocytes with 1% Triton X-100 in PBS and PBS alone, respectively.
Brine shrimp bioassay
Brine shrimps were hatched using brine shrimp eggs in a glass rectangular vessel (5 L), filled with sterile artificial seawater, prepared using water (2 L), NaCl (46 g), MgCl2 ·6H2O (22 g), Na2SO4(8 g), CaCl2 ·2H2O (2.6 g), and KCl (1.4 g), with a pH of 9.0 adjusted with Na2CO3, under constant aeration for 48 hr. After hatching, active nauplii free from egg shells were collected from the brighter portion of the hatching chamber and used for the assay. A hundred microliters of suspension of nauplii containing 10 larvae were added in all the wells of the 96-well microplate. Four jellyfish venoms (0.1-2 mg/ml) were added in each well and incubated at room temperature for 24 hr. The plates were then examined under a microscope (12.5×) and the number of dead nauplii in each well counted. Control experiments were performed with PBS in place of venom. The percentage lethality was determined by comparing the mean surviving larvae of the test and control wells. Lethal concentration (LC50) values were obtained from the best-fit line plotting concentration versus percentage lethality.
SDS-PAGE
Electrophoresis was carried out according to Laemmli method [13] using 12% polyacrylamide gel with 4% stacking gel. Samples were resuspended in SDS?PAGE sample buffer (62.5 mM Tris?HCl pH 6.8, 10% glycerol, 2% SDS, 0.01% bromophenol blue) and incubated at 95℃ for 5 min, then stored at ?20℃ until use. Each jellyfish venoms (25 μg) were electrophoresed for 90 min at 100 V constant voltage at room temperature, using Tris-glycine running buffer. The molecular weight size marker, 3.5-260 kDa (Novex Sharp pre-stained protein standards, Invitrogen, C.A. USA), was run parallel with venom sample for molecular weight estimation. Protein bands were visualized by Coomassie R-250.
Results
Cytotoxicity of four jellyfish species venoms
Heart myoblast H9C2 and skeletal myoblast C2C12 cells were incubated for 24 hr with various concentrations of four jellyfish species venoms. A comparison of relative cytotoxicity on the cells was shown in Fig. 1. H9C2 and C2C12 cells showed a venom concentration-dependent cell death and their calculated LC50 were displayed in Table 1. As shown, the level of cytotoxicity varied amongst the species with following rank order of potency, N. nomurai > C. nozakii > A. aurita > R. esculentum. Interestingly, venoms from N. nomurai, A. aurita and R. esculentum were more potent in the H9C2 cells compared to the C2C12 cells whereas venom from A. aurita was more potent in the C2C12 cell line (Table 1). Therefore, these results suggest that treatment with four jellyfish venoms can induce cytotoxicity against heart muscle and skeletal muscle with a higher potency in the heart.
Fig. 1.
Cytotoxicity of the four jellyfish venoms on (A) H9C2 and (B) C2C12 cells. Exponentially growing H9C2 (cardiac myoblast cell) and C2C12 (skeletal muscle cell) were treated with various concentrations of four jellyfish venoms for 24 hr. The cytotoxic effect was assessed by measuring mitochondrial dehydrogenase activity, using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The formazan crystal generated was dissolved by adding dimethyl sulfoxide (DMSO) and the absorbance was determined at 540 nm using a spectrophotometric microplate reader (BioTek Instruments, Inc., Winooski, USA). Cells incubated with no venom (PBS) were taken as proper controls. The data shown are the mean ± SD of three independent experiments.
Comparison of hemolytic activity of four jellyfish species venoms
Four jellyfish species venoms were assessed for its hemolytic activity using the blood samples of dog, human and rat. Difference in hemolytic activity between venoms detected at same venom concentration (1 mg/ml) in dog, human and rat erythrocytes. The venom of N. nomurai was found to be the most potently hemolytic and R. esculentum was the least hemolytically active (Fig. 2).
Fig. 2.
Comparison of hemolytic activity of the four jellyfish venoms in several species of erythrocytes. Freshly prepared erythrocytes from (A) dog, (B) human, and (C) rat were resuspended in PBS to make 1% solution and incubated with indicated concentration (1mg/ml) of each jellyfish venom for 30 min at 37℃ water bath. The mixtures were centrifuged at 1,500 × g for 5 min at 4℃, and the supernatants were transferred to 96-well microplates and the absorbance at 415 nm was determined using a spectrophotometric microplate reader to quantify the extent of red blood cell lysis. Positive control (100% hemolysis) and negative control (0% hemolysis) were determined by treating erythrocytes with 1% Triton X-100 (in PBS) or PBS alone, respectively. The data shown are the mean ± SD of three independent experiments.
Comparison of four jellyfish species venoms toxicity to brine shrimp
Results of the toxicity against brine shrimp of the venoms from four jellyfish species are shown in Fig. 3. The venom of all four jellyfish species (N. nomurai, C. nozakii, A. aurita and R. esculentum) caused significant toxicity against brine shrimp with LC50 values 52.31, 68.84, 249.70 and 762.50 μg/ml, respectively.
Fig. 3.
Comparison of the four jellyfish venoms toxicity to brine shrimp. One hundred microliters of sea salt water containing 10 adult brine shrimp was placed in all the wells of the 96-well microplate. Whole of four jellyfish venoms were added to the seawater/brine shrimp suspension and incubated for 24 h. Experiments were conducted along with control and different concentrations (0.1-1000 mg/ml) in a set of three wells per dose. The percentage lethality was determined by comparing the mean surviving larvae of the test and control wells. Lethal concentration (LC50) values were obtained from the best-fit line plotting concentration versus percentage lethality. The data shown are the mean ± SD of three independent experiments.
Comparison of SDS-PAGE pattern of four jellyfish species venoms
To characterize the protein components of four jellyfish species venoms, we separated the venoms proteins using SDS-PAGE. As shown in Fig. 4, all of the venoms contained numerous proteins with various sizes of molecular weight. The profile of four jellyfish venoms were very similar with major bands being located around 40 kDa. However, many different components were also located around this region demonstrating high variability among the venoms and several other weakly stained bands were observed in all venoms.
Fig. 4.
SDS-PAGE separation of four jellyfish venoms proteins. Electrophoresis was carried out according to Lammeli method using 12% polyacrylamide gel. Whole of four jellyfish venoms proteins (50 mg) were electrophoresed for 90 min at 100 V constant current in room temperature, using Tris-glycine running buffer. The molecular weight size marker, in the range of 10-260 kDa, was run parallel with venom sample for molecular weight estimation. Protein bands were visualized by staining gel with Coomassie R-250 dye. Lane 1, the molecular weight size marker; Lane 2, 3, 4 and 5, aliquot of jellyfish venom, C. nozakii, A. aurita, R. esculentum, and N. nomurai, respectively.
Discussion
Human envenomation is mainly due to the species like N. nomurai, C. nozakii, A. aurita and R. esculentum in Korea, China and Japan seas [2,4,14]. Although the important impact of jellyfish envenomation on public health has to be mentioned because of its consequences on humans, so that studies have been also carried out with the aim of preventing or reducing pathological effects deriving from jellyfish venoms. Therefore, comparative assessment of jellyfish venoms is the first step in furthering our understanding of jellyfish toxin's mode of action, which in turn should permit the development of more effective remedies against jellyfish stings. For these reasons, we performed comparative studies on four jellyfish species venoms using several experimental assays.
The cytotoxicity test to examine the potency of jellyfish venoms in H9C2 (heart myoblast) and C2C12 (skeletal myoblast) cells. Exposure to the jellyfish venoms resulted in concentration-dependent cell death with a much higher potency in H9C2 than C2C12, with the exception of C. nozakii venom (Fig. 1). This was supported by previous studies which demonstrated that jellyfish venoms showed selective toxicity on cardiac tissue [15, 16]. In addition, since the rank order potencies were the same for the two cell lines, it appears as though the cellular target is the same in both cell lines.
Hemolytic activity has been demonstrated in venoms from a number of box jellyfish, including Carybdea alata [17], Carybdea marsupialis [18], and Chironex fleckeri [19]. The presence of hemolytic activity in four species of jellyfish venoms against dog, human and rat erythrocytes also investigated. Interestingly, N. nomurai jellyfish venom demonstrated the greatest hemolytic potency, in consistent with its cytotoxicity (Figs. 1 and 2). Cytolytic toxins are known to operate by either of two general mechanisms. For enzymatic mechanism, cytolytic venoms of marine invertebrates bind preferentially to membrane glycolipids or glycoproteins [20]; for stoichiometric mechanism, it involves binding and insertion of toxin molecules into the plasma membrane followed by oligomerization to form transmembrane pores, and resulting colloid osmotic lysis [21]. It was thought that jellyfish venom formed pore like structures in target membranes causing rapid cell lysis [22]. The brine shrimp bioassay serves as a test organism in a wide range of toxicological assays and research. This bioassay has been demonstrated to provide a viable alternative to the mouse toxicity, which is expensive and associated with ethical constraints [23]. Being consistent with above results, N. nomurai venom caused most toxic against brine shrimp (Fig. 3).
For analysis of the protein components of four species jellyfish venoms, SDS-PAGE assay was used. The similar pattern with molecular weight of approximately 40 kDa was identified. Recently, cytolysin toxins (40-46 kDa) from box jellyfish (C. fleckeri, C. alata, and Carybdea rastonii) have been identified [17, 24, 25]. The proteins in the newly discovered family of box jellyfish toxins has been are particularly interesting because they are all potent cytolysin with the potential to be lethal and cause harmful effects in envenomed humans [26]. Consequently, we have now no evidence, however, whether some of these proteins contribute to the cytotoxicity, hemolytic activity and brine shrimp toxicity of these jellyfish venoms.
In conclusion, we first compared four species jellyfish nematocyst venom with regard to cytotoxicity, hemolytic activity, and brine shrimp toxicity. All of venoms had potent cytotoxic, hemolytic activities and brine shrimp toxicity, which might be attributed to the toxicologically active protein components of the venoms. However, to fully understand the mechanism of venom character is needed. Therefore, further biochemical and toxicological investigations will require to characterize the different active protein components of four jellyfish venoms for a toxicological barometer and to clarify their mechanism of action.
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