Cytotoxicity of the Nematocyst Venom from the Sea Anemone Aiptasia Mutabilis

Comparative Biochemistry and Physiology, Part C 139 (2004) 295–301 www.elsevier.com/locate/cbpc

Cytotoxicity of the nematocyst venom from the sea anemone Aiptasia mutabilis

Angela Marinoa, Vincenza Valverib, Carmelo Muia`a, Rosalia Crupib, Gianluca Rizzob, Giovanni Muscib,*, Giuseppa La Spadaa

aDepartment of Physiology and Pharmacology, University of Messina, Salita Sperone, 31-98166 Messina, Italy bDepartment of Microbiological, Genetic and Molecular Sciences, University of Messina, Salita Sperone, 31-98166 Messina, Italy

Received 1 September 2004; received in revised form 3 December 2004; accepted 12 December 2004

Abstract

Crude extracts of the coelenterate Aiptasia mutabilis (Anthozoa, Aiptasiidae) nematocysts have been tested for their cytotoxicity of Vero and HEp-2 cells monolayers. The results indicate that the nematocyte venom contains one or more toxins with an extremely powerful cytolytic activity. An extract containing the equivalent of as little as 0.6 nematocysts/AL is sufficient to induce significant cellular necrosis, and IC50 can be estimated to be ca. 2 nematocysts/AL on Vero cells. These values are 1–2 orders of magnitude lower than those reported so far for other sea anemone venoms. The extreme potency is accompanied by poor stability of the venom that is readily inactivated by moderate heat and by buffers at non-neutral pH values. The extract is unstable even when kept for short times at 4 8C, or after storage at À20 8C. Separation of crude venom by affinity chromatography on ConA-Sepharose allowed us to identify two main components with molecular masses of 95 and 31 kDa, respectively, as responsible for the cytolytic properties of A. mutabilis nematocyst extract. D 2004 Elsevier Inc. All rights reserved.

Keywords: Coelenterate toxins; Nematocysts; Aiptasia

1. Introduction investigations have been also performed with the aim of preventing or reducing pathological effects deriving from One of the most relevant aspects of cnidarian physiology cnidarian toxins. Immunological studies have been also is related to the biologically active compounds of nemato- carried out trying to detect antibody titers against nematocysts, organelles contained in specialized cells called cysts venom (Radwan et al., 2000). nematocytes. The accidental touch with some Coelenterate Cnidarians, with special regard to sea anemones, specimens can produce severe local and systemic patholo- generally contain one or more cytolytic peptides or proteins, gies, and, in some case, can lead to death. but it is usually difficult to localize such bioactive Human pathologies are mainly due to specimens like compounds in nematocysts or even in intact tissues, as in Physalia physalis, Chironex fleckeri and Pelagia noctiluca. tentacles or in achroragi. In some species, as Hydra However, the large variety of potentially toxic species and vulgaris, toxins are stored inside the nematocyst (Klug et the different extraction techniques employed to obtain crude al., 1989). Toxins may also be secreted by intact contracting venom have prevented so far an exhaustive description of sea anemones on mechanical stimulation (Sencic and nematocyte toxinological features. Nevertheless the notable Macek, 1990). impact of cnidarian envenomation on public health has to be Bioactive compounds of nematocysts include proteins mentioned because of its consequences on humans, so that (neurotoxins, pore-forming toxins or cytolysins, phospholi- pases, proteinase inhibitors) and secondary metabolites with * Corresponding author. Tel.: +39 906765194; fax: +39 90392733. either toxic and/or biomedical properties. Little is known on E-mail address: [email protected] (G. Musci). the mechanism of action of nematocyst toxins. Delay in the

+ voltage-dependent Na channels inactivation has been 10 mM; MgCl2, 24 mM; MgSO4, 28 mM; imidazole, 5 mM; p reported, however most toxic effects of cnidarian enveno- pH=7.65, =1100 mOsm/kgH2O), then collected in a tube mation seem to derive from impaired membrane perme- and treated with a 1 M sodium citrate solution. After 10 min ability. In particular, membrane permeability can be affected of incubation, acontia were gently pipetted to allow by pore-forming toxins: in some cases these proteins have nematocysts detachment and the isolated capsules were been sequenced and even cloned (Anderluh et al., 1999)to then repeatedly washed in ASW. Isolated nematocysts be then employed as model proteins to study protein–lipid (mostly belonging to the microbasic-mastigophore type) membrane interactions. were either immediately used or stored at À20 8C. Stored Many biological assays have been carried out to evaluate capsules were utilized when A. mutabilis specimens were toxicity of venoms. Among the most used is the haemolytic unavailable due to bad environmental conditions. Frozen assay on red blood cells from different sources (Chung et nematocysts were rinsed with ASW after thawing. They al., 2001; Lanio et al., 2003; Marino et al., 2004; were anatomically intact and functionally competent as Monastyrnaya et al., 2002). Neurotoxic (Gondran et al., shown by their full discharge capability. 2002), cardiotoxic (Bruhn et al., 2001) or cytolytic tests (Anderluh and Macek, 2002) are also employed. Recently, 2.3. Cell cultures evaluation of cytotoxic activities has been performed on V79 cells with toxins of Rhizostoma pulmo (Allavena et al., Monolayer renal monkey Vero or human epithelial HEp- 1998), P. noctiluca (Mariottini et al., 2002) and of 2 cell lines (American Type Culture Collection) were Anemonia sulcata and Aequorea aequorea (Carli et al., employed for cytolytic assays. Cells were cultured in 1996). On the other hand, toxins of P. physalis have been Dulbecco’s modified Eagle’s medium or in RPMI 1640 tested on L-929 cells (Edwards et al., 2002), a mouse supplemented with 10% fetal bovine serum (FCS), 2 mM l- fibroblast cell line. Interestingly some recent observations glutamine, 100 U/mL penicillin, 100 Ag/mL streptomycin at show that Cnidarian venoms, as that from Chiropsalmus 37 8Cwith5%CO2. Cell density of each culture was quadrigatus, can even induce apoptosis in glioma and assessed before the experimental treatment by counting in vascular endothelial cell line (Sun et al., 2002). Moreover, Burker chamber. Kikuchi and colleagues (Kikuchi et al., 1982, 1983) have identified clavulone, a compound from Clavularia viridis, 2.4. Venom preparation which has been later shown to be able to inhibit tumor cell growth in leukemia HL-60 (Honda et al., 1985). Fresh or thawed nematocysts were pelleted, resuspended It has to be pointed out that toxins are often extracted to a density of about 6Â104 nematocysts/mL and sonicated from whole animals or from excised tentacles but not from on ice with a Sonoplus (70 MHz, 20 s, 20 times) in RPMI isolated nematocysts, so that their presence in the capsular 1640, unless otherwise stated. Nematocysts integrity and fluid should be considered as putative. The aim of this work functionality was not affected by RPMI 1640 as indicated was to verify and characterize the in vitro cytotoxicity of by proper control experiments. The crude extract was crude extracts from isolated microbasic-mastigophore nem- separated from crushed nematocysts by centrifugation atocysts of Aiptasia mutabilis (Anthozoa, Aiptasiidae) from (refrigerated centrifuge ALC PK 120 R, 3000Âg, 5 min) the Strait of Messina. and the supernatant was diluted to different doses with RPMI 1640 before running the cytolytic assay. Protein concentration of the extract was measured by the bicincho- 2. Materials and methods ninic acid assay (Pierce). Crude venom was diluted 10–1000 fold before use, corresponding to a protein content ranging 2.1. Materials from 1.7Â10À3 Ag/AL to 1.7Â10À1 Ag/AL.

All reagent grade chemicals were from Sigma Italia, 2.5. Cytolytic assays Milan. Crude venom was diluted to the proper concentration 2.2. Nematocysts isolation with RPMI 1640 and dispensed in microplate wells containing cultured cells. Cell necrosis was verified by Specimens of A. mutabilis were collected in summer inverted microscope observations after 1 h of incubation at time in the Strait of Messina along the coasts of Calabria 37 8C with 5% CO2, by using Trypan blue test. In particular, and Sicily (Italy), maintained in closed circuit aquaria at 18– cells were trypsinized, washed with phosphate buffered 22 8C and weekly fed with shrimp. saline (PBS) and treated with Trypan blue solution. Necrotic Nematocysts were isolated as previously described cells were then counted among a significant population (at (Marino et al., 2004): briefly, acontia were excised from least 400 cells). the specimens, washed several times in low Ca2+-artificial The percentage of apoptotic cells was calculated after sea water (ASW: NaCl, 520 mM; KCl, 9.7 mM; CaCl2, acridine orange staining. Briefly, Vero cells treated with A. A. Marino et al. / Comparative Biochemistry and Physiology, Part C 139 (2004) 295–301 297

A mutabilis crude venom were re-seeded in multiwell glasses ° pretreated with 0.1 mg/mL polylysine, fixed with Carnoy’s 100 10 min solution for 15 s and then dehydrated with decreasing ° 30 min concentrations (95–30%) of ethanol. Glasses were then 75 60 min rapidly dipped in 1% citric acid, washed in distilled water ° and immersed in citric acid/sodium phosphate McIlvaine 50 buffer for 5 min. Cells were finally stained with 0.01% ° acridine orange in McIlvaine buffer for 5 min, washed in % necrosis PBS and observed by epifluorescence microscopy. At least 25 * 400 total cells were counted to estimate percent of * apoptosis. 0 1:10 1:30 1:100 1:1000 2.6. Miscellaneous venom dilution B Denaturing SDS–PAGE on 7.5% polyacrylamide gels 100 was performed according to Laemmli (1970). Molecular ° weight markers were from Sigma Italia, Milan. Chromatography of crude venom on ConA-Sepharose 75 was performed according to standard procedures. The resin was equilibrated in 0.01 M phosphate buffer containing 50 ° 0.9% NaCl. After passage of crude venom, the resin was washed with 10 volumes of the same buffer (collected in 1 % necrosis mL fractions) and bound proteins were eluted with two 25 volumes of phosphate buffer containing NaCl 1 M and a- methylmannoside 1 M at room temperature for 60 min, 0 repeated twice. Eluted fractions were dialysed before use. 1:10 1:30 1:100 1:1000 Unless otherwise stated, data were expressed as mean- venom dilution FS.E. from three separate experiments. One-way ANOVA Fig. 2. Cytolytic effect of Aiptasia mutabilis crude venom on Vero (panel followed by Dunnett’s test was used to determine the A) and HEp-2 (panel B) cells. The venom was tested at different dilutions significant differences. GraphPad Prism was used to and (for Vero cells) at different incubation times. Bars represent the perform statistical analyses. meanFS.E. from three separate experiments. Cells incubated for the specified times with no venom were taken as proper controls. One-way ANOVA followed by Dunnett’s test was used to determine the significant differences: *Pb0.05 vs. control. 8Pb0.01 vs. control. 3. Results

3.1. Characterization of A. mutabilis crude venom components with molecular weights of approximately 95, 52 and 31 kDa were present. Several other bands could be The SDS–PAGE pattern of a fresh extract of A. mutabilis discerned, most of them being comprised in the 35–55 kDa crude venom is reported in Fig. 1, panel A. Three major range of molecular weight.

AB3.2. Cytotoxicity of A. mutabilis crude venom HMW LMW venom kDa LMW 1 2 3 4 Vero and HEp-2 cells were incubated for different times, 200 kDa 116 namely 10, 30 and 60 min, with different amounts of A. 97 97 mutabilis crude venom. After the established times, cells 66 66 were analyzed for their survival. Fig. 2A shows the percent 45 45 of necrosis of Vero cells under these conditions. As shown, a 10-fold dilution of the nematocyst extract, corresponding 31 31 to a protein concentration of ca. 0.17 mg/mL, was sufficient to induce an essentially complete necrosis of the cell layer 21 21 after 1 h. The response, however, was also significant at shorter times and/or at lower doses. As a matter of fact, a Fig. 1. SDS-PAGE analysis of Aiptasia mutabilis crude venom. Panel A significant effect ( pb0.05) was elicited by the lowest shows a freshly prepared nematocyst extract. Panel B shows the various fractions obtained after affinity chromatography on ConA-Sepharose. Lane dilution after an incubation time as short as 10 min. On 1, unretained fraction; lane B, PBS washing, lanes 3 and 4, fractions eluted the other hand, the necrotic effect of a 100-fold dilution, with a-methylmannoside. corresponding to a protein concentration of 17 Ag/mL, 298 A. Marino et al. / Comparative Biochemistry and Physiology, Part C 139 (2004) 295–301 turned out to be statistically significant ( pb0.05) after 1 h of A 100 incubation. HEp-2 cells displayed an essentially similar control behavior, as shown in Fig. 2B for the 60 min long liophylized 75 incubation time. As observed with Vero cells, the effect -80°C was linearly dependent on venom concentration. However, -20°C HEp-2 cells were slightly less susceptible to A. mutabilis 50 toxins, since a complete cell necrosis was never observed even at the highest dose tested. Consistently, a lower % necrosis dilution had to be used to achieve a statistically significant 25 effect. ° * To investigate whether apoptosis played a role in the 0 observed cytotoxic effects of A. mutabilis crude venom, 1:10 1:30 1:100 1:1000 cells were treated with various concentrations of the venom dilution B nematocyst extract and stained with acridine orange at 100 different times of treatment. No significant staining was control observed under any experimental condition. liophylized 75 A partial biochemical characterization of the toxin(s) -80°C responsible for the cytolytic effects was carried out. ° ° -20°C Chromatography on ConA-Sepharose was performed in 50 order to assess the glycoproteic nature of the toxin(s). Fig. 1, panel B, reports the SDS patterns of the unretained fraction % necrosis (lane 1), of the PBS washing (lane 2) and of the two 25 * fractions eluted with a-methylmannoside (lanes 3 and 4). ° ° Clearly, two main species with molecular weights of 95 and 0 31 kDa were retained by ConA-Sepharose. Cytolytic tests 1:10 1:30 1:100 1:1000 were run on the various fractions and revealed that the venom dilution components eluted with the sugar had very strong cytolytic Fig. 3. Effect of different storage conditions on the cytolytic potency on activity; the unretained fraction and the PBS washing, on Vero (panel A) and HEp-2 (panel B) cells of Aiptasia mutabilis crude the other hand, displayed poor cytolytic power. venom. The venom was tested at various dilutions after the indicated storage treatments (see text for details). The venom tested immediately after 3.3. Stability of the crude venom extraction from nematocysts served as control. Bars represent the mean- FS.E. from three separate experiments. One-way ANOVA followed by Dunnett’s test was used to determine the significant differences: *Pb0.05 In order to assess the stability to storage of the crude vs. control. 8Pb0.01 vs. control. extract of A. mutabilis nematocysts, samples of the venom were tested for their cytotoxicity just after extraction, and after storage for 5 days at À80 8C, À20 8C or in the the second dilution tested (1:30). With both Vero and HEp-2 lyophilized state. The results on both Vero and HEp-2 cells cells, higher dilutions gave values of necrosis too low to be are reported in Fig. 3. Clearly, storage at the lowest properly analysed. The lyophilization process did not temperature did not impair the cytotoxic ability of the crude grossly affect the molecular weights of the proteic compo- venom at any tested dilution, with the notable exception of nents of the venom mixture, since the pattern of a sample the lowest dilution tested on HEp-2 cells. On the other hand, lyophilized and then redissolved to the starting volume was storage at À20 8C greatly and significantly reduced the undistinguishable from that of a fresh extract (data not biological activity of the extract, and essentially no necrosis shown). In particular, no aggregates were evident in the was induced even at the highest venom concentration tested. sample subjected to lyophilization. The results obtained with samples of venom redissolved The crude venom was also tested for its thermal stability, after lyophilization were more complex, as they were and the results of a representative series on Vero cells are apparently dilution- as well as cell type-dependent. In the shown in Fig. 4. For this experiment, the extract was case of Vero cells, lyophilization did not affect the toxin incubated at different temperatures (4 8C, 20 8C, 40 8C and potency when tested at the lower dilution, while it 60 8C) for different times (5, 30 and 60 min) before being apparently impaired the cytotoxic activity of a more diluted tested for cytotoxicity on the monolayer. As expected for a (i.e., 1:30) sample (Fig. 3A). It should be noted, however, proteic extract, the stability was progressively affected by that the statistical analysis revealed that even in this case the higher temperatures and incubation times. In general, it is difference was not significant ( pN0.05). HEp-2 cells were quite clear from data in Fig. 4 that the extract of A. mutabilis significantly ( pb0.01) less responsive to lyophilized–redis- nematocysts is highly unstable. The cytolytic potency of the solved extracts even at the highest dose (Fig. 3B). The extract was significantly impaired even after 1 h at 4 8C, difference in potency remained significant ( pb0.05) even at with the effect being more pronounced at higher dilutions. A. Marino et al. / Comparative Biochemistry and Physiology, Part C 139 (2004) 295–301 299

100 100 4°C 5 min 20°C 30 min 75 75 60 min

50 50 % necrosis % necrosis 25 25

0 0 1:10 1:30 1:100 1:10 1:30 1:100 venom dilution venom dilution 100 100 40°C 60°C 75 75

50 50 % necrosis 25 % necrosis 25

0 0 1:10 1:30 1:100 1:10 1:30 1:100 venom dilution venom dilution

Fig. 4. Thermostability of Aiptasia mutabilis crude venom. The nematocyst extract was incubated at the indicated temperatures (4 8C, 20 8C, 40 8C, 60 8C) for the indicated times (5, 30, 60 min) and then tested on Vero cells. Cells were incubated with the temperature-treated venom for 1 h. The graph shows the results from a representative experiment.

Not much difference was observed when the extract was medium used during venom preparation was brought to kept at 20 8C. On the other hand, a great loss of cytotoxicity proper pH by addition of 0.5 M acetate (pH 4.5, 5.5), 0.1 M was observed at higher temperatures, as shown by graph phosphate (pH 6.5, 7.5, 8.5) or borate buffers (pH 9.5). reporting data at 40 8C and 60 8C. At this latter temperature After 15 min of incubation at the various pH values, the the cytotoxic activity of the venom essentially collapsed extract was diluted as usual before being tested on a Vero after incubation of the extract for as short as 10 min. cells monolayer. The results, shown in Fig. 5, suggest that the optimal pH stability of the extract is around pH 7.5. 3.4. pH dependence of venom stability

The effect of pH on the cytotoxic activity of A. mutabilis 4. Discussion nematocyst extract was investigated. At this purpose, the The results presented in this paper unequivocally show 100 that A. mutabilis nematocysts contain one or more powerful toxins, with a strong cytotoxic activity. This is consistent with previous data in the literature, showing that Cnidaria 75 toxins are stored both in nematocytes and in surrounding tissues (Endean and Noble, 1971; Wittle et al., 1971). As 50 little as 600 nematocysts per mL, corresponding to 1.7Â10À2 mg of protein per mL of venom solution are sufficient to elicit a significant effect on the cultured Vero % necrosis 25 and HEp-2 monolayer, although these latter cells were slightly less sensitive. In terms of IC50 expressed as 0 nematocyst concentration, data in Fig. 2 show that a 1:30 4.5 5.5 6.5 7.5 8.5 9.5 dilution of our venom preparation exerts a ca. 50% mortality pH effect after 1 h on Vero cells, corresponding to an IC50 value of 2000 nematocyst per mL. This suggests an extremely Fig. 5. pH-resistance of Aiptasia mutabilis crude venom. The nematocyst extract was incubated at different pH values for 1 h and then tested on Vero high toxicity of A. mutabilis venom, since previous studies cells. Acetate, phosphate and borate buffers were used to attain the proper on toxicity of jellyfish and sea-anemone venoms, namely pH values. The graph shows the result from a representative experiment. from P. noctiluca (Mariottini et al., 2002) and from A. 300 A. Marino et al. / Comparative Biochemistry and Physiology, Part C 139 (2004) 295–301 aequorea, R. pulmo and A. sulcata (Carli et al., 1996) have mutabilis crude venom shows a major band at around 95 reported IC50 values between 39,900 and 76,600 nemato- kDa, a Mr value closely corresponding to that of the co-lytic cysts per mL, with even longer incubation times in some factor identified in A. pallida by Grotendorst and Hessinger cases. (1999), and that this component is retained in the active The effect seems to be necrotic in nature, and no fraction after ConA-Sepharose (see Fig. 1B). On the other evidence for a toxin-induced apoptotic process has been hand, A. pallida PLA2, has a molecular weight higher than. obtained. In particular, staining with acridine orange ruled 40 kDa, while the active species found in A. mutabilis crude out that apoptosis was taking place under any experimental venom has an apparent weight of 31 kDa. condition (venom dilution, incubation time). In conclusion, our results confirm that crude venom from The toxins are most likely of proteic nature, as indicated sea-anemones belonging to the genus Aiptasia is strongly by their thermal instability. As a matter of fact, even short cytotoxic. Further biochemical investigations are in progress incubations at 40 8C are sufficient to greatly reduce the to characterize the different proteic components of A. cytotoxic potency of the venom. The storage stability assays mutabilis nematocysts extract from a physico-chemical show that even at 4 8C the venom is unstable. At low viewpoint and to elucidate their mechanism of action. concentrations, 1 h is sufficient to significantly moderate the cytotoxic effect, and 24 h at 48 lower to less than 15% the cytotoxic properties of the venom even at high protein Acknowledgement concentrations (data not shown). The concentration-dependence of the venom potency can be deduced by both the We wish to thank Professor A. Mastino for providing us storage and the temperature-dependence experiments. In Vero and HEp-2 cell lines. both cases, diluted samples of the nematocyst extract seem to be more prone to degradation. In this respect, it is interesting to note that the lyophilization process probably References affects the toxin structure in a bborderlineQ fashion, and that the diluted state synergistically act to denature and Allavena, A., Mariottini, G.L., Carli, A.M., Contini, S., Martelli, A., 1998. inactivate them (cf. Fig. 3). In vitro evaluation of the cytotoxic, hemolytic and clastogenic activities Separation of the crude venom by means of ConA- of Rhizostoma pulmo toxin(s). Toxicon 36, 933–936. Sepharose yielded a clear-cut result, in that two species with Anderluh, G., Macek, P., 2002. Cytolytic peptide and protein toxins from sea anemones (Anthozoa: Actiniaria). Toxicon 40, 111–124. molecular weights of 95 and 31 kDa were clearly isolated Anderluh, G., Barlic, A., Podlesek, Z., Macek, P., Pungercar, J., and turned out to be mainly responsible for the cytotoxic Gubensek, F., Zecchini, M.L., Serra, M.D., Menestrina, G., 1999. properties of A. mutabilis nematocyst extract. While we Cysteine-scanning mutagenesis of an eukaryotic pore-forming toxin cannot rule out, at this stage, the possibility that other from sea anemone: topology in lipid membranes. Eur. J. Biochem. species contribute to the cytolytic power of the crude 263, 128–136. Bruhn, T., Schaller, C., Schulze, C., Sanchez-Rodriguez, J., Dannmeier, C., venom, the chromatographic result strongly suggests that Ravens, U., Heubach, J.F., Eckhardt, K., Schmidtmayer, J., Schmidt, the 31 kDa component, which is present at high concen- H., Aneiros, A., Wachter, E., Beress, L., 2001. Isolation and character- tration in both fractions eluted with a-methylmannoside, is isation of five neurotoxic and cardiotoxic polypeptides from the sea the main glycotoxin present in A. mutabilis nematocysts. anemone Anthopleura elegantissima. Toxicon 39, 693–702. So far, only very few toxins have been proven to be Calton, G.J., Burnett, J.W., 1988. Characterization of nematocyst venoms. Nematocyst venoms and toxins. In: Hessinger, D.A., located in nematocysts (Calton and Burnett, 1988; Groten- Lenhoff, H.M. (Eds.), The Biology of Nematocysts. Academic Press, dorst and Hessinger, 1999; Lotan et al., 1996). Among San Diego, pp. 369–374. these, a phospholipase A2 (PLA2) activity, an essential Carli, A., Bussotti, S., Mariottini, G.L., Robbiano, L., 1996. Toxicity of component of the cytolytic systems of snake (Roy, 1945) jellyfish and sea-anemone venoms on cultured V79 cells. Toxicon 34, and bee (Habermann and Kowallek, 1970), has been found 496–500. Chung, J.J., Ratnapala, L.A., Cooke, I.M., Yanagihara, A.A., 2001. Partial in the nematocysts of the sea anemone Aiptasia pallida purification and characterization of a hemolysin (CAH1) from Hawaiian (Grotendorst and Hessinger, 1999; Hessinger and Lenhoff, box jellyfish (Carybdea alata) venom. Toxicon 39, 981–990. 1976), a species closely related to the object of our Edwards, L.P., Whitter, E., Hessinger, D.A., 2002. Apparent membrane investigation. It should be noted that the venom of A. pore-formation by Portuguese Man-of-war (Physalia physalis) venom pallida has been proven so far to be cytolytic only to red in intact cultured cells. Toxicon 40, 1299–1305. Endean, R., Noble, M., 1971. Toxic material from the tentacles of the blood cells (Hessinger and Lenhoff, 1973). Our data show cubomedusan Chironex fleckeri. Toxicon 9, 255–264. Aiptasia venoms can have a wider cytolytic activity, being Gondran, M., Eckeli, A.L., Migues, P.V., Gabilan, N.H., Rodrigues, A.L., cytotoxic against a variety of cell types. It is possible that a 2002. The crude extract from the sea anemone, Bunodosoma caissarum elicits convulsions in mice: possible involvement of the glutamatergic PLA2 component is involved in A. mutabilis toxicity, since the parental enzyme in A. pallida is a glycoprotein readily system. Toxicon 40, 1667–1674. Grotendorst, G.R., Hessinger, D.A., 1999. Purification and partial inactivated at temperature above 40 8C and has a pH characterization of the phospholipase A2 and co-lytic factor from optimum of 7.7 (Grotendorst and Hessinger, 2000). It is sea anemone (Aiptasia pallida) nematocyst venom. Toxicon 37, interesting to point out that the electrophoretic pattern of A. 1779–1796. A. Marino et al. / Comparative Biochemistry and Physiology, Part C 139 (2004) 295–301 301

Grotendorst, G.R., Hessinger, D.A., 2000. Enzymatic characterization of sodium dodecyl sulfate on the conformation and hemolytic activity of the major phospholipase A2 component of sea anemone (Aiptasia St I and St II, two isotoxins purified from Stichodactyla helianthus. pallida) nematocyst venom. Toxicon 38, 931–943. Toxicon 41, 65–70. Habermann, E., Kowallek, H., 1970. Modifications of amino groups and Lotan, A., Fishman, L., Zlotkin, E., 1996. Toxin compartmentation and tryptophan in melittin as an aid to recognition of structure–activity delivery in the Cnidaria: the nematocyst’s tubule as a multiheaded relationships. Hoppe-Seyler Z. Physiol. Chem. 351, 884–890. poisonous arrow. J. Exp. Zool. 275, 444–451. Hessinger, D.A., Lenhoff, H.M., 1973. Binding of active and inactive Marino, A., Musci, G., La Spada, G., 2004. Hemolytic effects of crude hemolytic factor of sea anemone nematocyst venom to red blood cells. venom from Aiptasia mutabilis nematocysts. Chem. Ecol. 20 (Suppl. Biochem. Biophys. Res. Commun. 53, 475–481. 1), S451–S459. Hessinger, D.A., Lenhoff, H.M., 1976. Membrane structure and function. Mariottini, G.L., Sottofattori, E., Mazzei, M., Robbiano, L., Carli, A., 2002. Mechanism of hemolysis induced by nematocyst venom: roles of Cytotoxicity of the venom of Pelagia noctiluca Forskal (Cnidaria: phospholipase A and direct lytic factor. Arch. Biochem. Biophys. 173, Scyphozoa). Toxicon 40, 695–698. 603–613. Monastyrnaya, M.M., Zykova, T.A., Apalikova, O.V., Shwets, T.V., Honda, A., Yamamoto, Y., Mori, Y., Yamada, Y., Kikuchi, H., 1985. Kozlovskaya, E.P., 2002. Biologically active polypeptides from the Antileukemic effect of coral-prostanoids clavulones from the stolonifer tropical sea anemone Radianthus macrodactylus. Toxicon 40, Clavularia viridis on human myeloid leukemia (HL-60) cells. Biochem. 1197–1217. Biophys. Res. Commun. 130, 515–523. Radwan, F.F., Gershwin, L., Burnett, J.W., 2000. Toxinological studies on Kikuchi, H., Tsukitani, Y., Iguchi, K., Yanada, Y., 1982. Clavulones, new the nematocyst venom of Chrysaora achlyos. Toxicon 38, 1581–1591. type of prostanoids from the stolonifer Clavularia viridis Quoy and Roy, A.C., 1945. Lecithin and venom haemolysis. Nature 155, 696. Gaimard. Tetrahedron Lett. 23, 5171–5174. Sencic, L., Macek, P., 1990. New method for isolation of venom from the Kikuchi, H., Tsukitani, Y., Iguchi, K., Yamada, Y., 1983. Absolute sea anemone Actinia cari. Purification and characterization of cytolytic stereochemistry of new prostanoids clavulone I, II and III from toxins. Comp. Biochem. Physiol., B 97, 687–693. Clavularia viridis Quoy and Gaimard. Tetrahedron Lett. 24, 1549–1552. Sun, L.K., Yoshii, Y., Hyodo, A., Tsurushima, H., Saito, A., Harakuni, T., Klug, M., Weber, J., Tardent, P., 1989. Hemolytic and toxic properties of Li, Y.P., Nozaki, M., Morine, N., 2002. Apoptosis induced by box Hydra attenuata nematocysts. Toxicon 27, 325–339. jellyfish (Chiropsalmus quadrigatus) toxin in glioma and vascular Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly endothelial cell lines. Toxicon 40, 441–446. of the head of bacteriophage T4. Nature 227, 680–685. Wittle, L.W., Middlebrook, R.E., Lane, C.E., 1971. Isolation and partial Lanio, M.E., Alvarez, C., Pazos, F., Martinez, D., Martinez, Y., purification of a toxin from Millepora alcicornis. Toxicon 9, 327–331. Casallanovo, F., Abuin, E., Schreier, S., Lissi, E., 2003. Effects of