- Email: [email protected]
Toxicity of jellyfish and sea-anemone venoms on cultured V79 cells
Copyright
0041-0101(95)00157-3
0
Toxicon. Vol. 34, No. 4, pp. 496-330, 1996 1996 Elsevier seiclla Ltd. All ri&s reserved Printed in Greal Britain co41~101/% SIS.00 + 0.00
TOXICITY OF JELLYFISH AND SEA-ANEMONE VENOMS ON CULTURED V79 CELLS A. CARLI,‘*
S. BUSSOTTI,’
G. L. MARIOTTINI’
and L. ROBBIANO*
‘Istituto di Scienze Ambientali Marine, Cattedra di Planctologia, Universiti di Genova, Viale Benedetto XV 5, 16132 Genova, Italy and ‘Istituto di Farmacologia, UniversS di Genova, Viale Benedetto XV 2, 16132 Genova, Italy (Received 9 August 1995; accepted 22 November 1995)
A. Carli, S. Bussotti, G. L. Mariottini and L. Robbiano. Toxicity of jellyfish and sea-anemone venoms on cultured V79 cells. Toxicon 34, 496-500, 1996.-Cnidarian toxins exert an influence on human activities and public health. The cytotoxicity of crude toxins (nematocyst and surrounding tissue venom) of Aequorea aequorea, Rhizostoma pulmo and Anemonia sulcata was assessed on V79 cells. Rhizostoma pulmo and Anemonia sulcata crude venoms showed remarkable cytotoxicity and killed all treated cells at highest tested concentration within 2 and 3 hr, respectively. Aequorea aequorea crude venom greatly affected growth rate during long-term experiments. No genotoxic effect was observed. Copyright 0 1996 Elsevier Science Ltd.
Cnidarian toxications are a world-wide health problem and can seriously affect some working (fishery) and tourist (bathing) activities. Some studies concerning Mediterranean Cnidaria led to toxin characterization of Actinia equina (Macek and Lebez, 1988), Anemonia sulcata (BCress et al., 1975), and partly of Pelagia noctiluca (Quadrifoglio et al., 1986; Salleo et al., 1986) and Rhizostoma pulmo (Cariello et al., 1988; Mazzei et al., 1994, 1995). Actually, excluding haemolytic effects, the cell-damaging action of Cnidarian toxins is greatly unknown (Bernheimer and Avigad, 1976; Giraldi et al., 1976; Batista et al., 1986, 1990; Batista and Jezernik, 1992; Mariottini et al., 1993; Allavena et al., 1995). Taking into account that toxins are located both in nematocysts and in surrounding tissues (Mar&al, 1974), the aim of this investigation was to determine the cytotoxicity in vitro of crude extracts obtained from nematocysts and tissues of Aequorea aequorea, Rhizostoma pulmo and Anemonia sulcata. Jelly&h A. aequorea (Hydrozoa) and R. pulmo (Scyphozoa) were collected in the Ligurian Sea and in the Northern Tyrrhenian Sea along the Eastern Sardinian coast, and kept at - 20°C until utilization. Sea-anemones A. sulcata (Anthozoa), were collected along the cliff of Sori (Ligurian Sea) and the coast of Porto Cervo (Northern Tyrrhenian * Author to whom correspondence should be addressed. 496
Short Communications
497
Sea), and were indefinitely maintained in aquaria. Tentacles of A. aequorea and A. sulcata and oral arm parts of R. pulmo were treated as reported (Arillo et al., 1994; Mazzei et al., 1994). Three extract amounts per species were prepared in relation to the counted nematocyst number (N), used as an estimate of toxin rate assuming that most of toxin is stored in nematocysts (dose A: 150,000 N/ml; dose B: 30,000 N/ml; dose C: 15,000 N/ml) and tested on V79 fibroblasts (NIH, Coriell Institute for Medical Research, Camden, NJ) for 1, 2 and 3 hr, and 5 days. Cell viability was evaluated both by Trypan Blue dye exclusion (TB; short- and long-term cytotoxicity) and neutral red assay (NR; according to Babich and Borenfreund, 1987; short-term citotoxicity). Cell morphology, detachment, colony forming efficiency (CFE) and growth rate (number of doublings) were also considered. The DNA fragmentation (single-strand breaks and/or alkali-labile sites) was evaluated by the alkaline elution technique (Brambilla et al., 1988). At the experimental temperature (37°C) no significant pH shifting from normal values was induced in culture medium by crude toxins. After 1 hr treatment with doses A and B of R. pulmo and dose A of A. sulcata cells appeared rounded and devoid of their typical extensions, several of them were detached from culture flasks. The cells treated with other doses of R. pulmo and A. sulcata and with all doses of A. aequorea showed usual morphology. After 2 hr cells also treated with dose B of A. sulcata showed the above reported morphological changes. No attached cells treated with dose A of R. pulmo and A. sulcata were observed after 3 hr, while all doses C and the dose B of A. aequorea did not appear different from control. Figure 1 shows short- and long-term cytotoxicity assessed by TB. The dose A of R. pulmo and A. sulcata induced fast toxic response, produced a noticeable decrease of cell viability after 1 hr and killed all cells after 2 and 3 hr, respectively. For A. aequorea dose A produced high cytotoxicity only after 3 hr. High toxic effects of dose B within the first hour and substantially no further toxicity were noticed by utilizing R. pulmo extracts, while for A. sulcata the effects were evident only after 3 hr. The dose C of the anemone produced slower effects than in R. pulmo. For A. aequorea data show a scarce influence of dose B on cell viability with effectiveness during the third hour, and a similar trend of doses A and C. During long-term experiments also the dose B of A. sulcata killed all cells and the dose C was highly toxic. Doses B and C of R. pulmo showed a remarkable toxicity. The toxin of A. aequorea allowed cell survival also with dose A. Overall, the toxicity of A. aequorea was similar at all doses and highly affected cell growth rate, as showed by calculated doublings; a slow growth rate in comparison with control was observed also using crude extracts of R. pulmo and A. sulcata (Carli et al., in press). NR results after 3 hr of treatment (Table la) are similar to those obtained using TB: lcso and IQ,, showed a strong cytotoxicity of R. pulmo and A. sulcata crude toxins and lower effects of A. aequorea. CFE was evaluated only at doses that permitted cell survival after the long-term experiment; preliminary results showed the dose C of R. pulmo allowed high CFE (75.5%; within normal values for V79 cells); values close to normality were observed also using dose B of R. pulmo (62.5%), doses B (64.0%) and C (65.0%) of A. aequorea, and dose C of A. sulcata (69.5%); on the contrary, dose A of A. aequorea showed remarkable effects (35.0%). Data provided by DNA damage/alkaline elution (Table lb) indicate that 1 hr treatment with subtoxic concentration of nematocysts of the three Cnidarian species did not cause increase of DNA elution rate, that might indicate the occurrence of DNA fragmentation. Under identical experimental conditions, a high frequency of DNA breaks was induced by 1 hr of exposure to 0.3 mM N-nitroso-N-methylurea used as positive control.
498
Short Communications
1 hour
2 hours
1 hour
2 hours
3 hours
100 90
80 0
70
S 2
60
%
50
B
40
E
30 20 10 0 3 hours
5 days
T 100
l
90
-.
80
-.
70
-.
60
..
50
..
40
-.
30
..
20
-.
C
10 -.
To
1 hour
2 hours
3 hours
5 days
timr
Fig. 1. Per cent survival in relation to controls of V79 cells treated with crude toxins extracted from A. aequorea, R. pulmo and A. sulcata during short- (1, 2 and 3 hr) and long-term (5 days) experiments (means and S.D. are calculated on four samples): (a) dose A (150,000 nematocysts/ml); (b) dose B (30,000 nematocysts/ml); (c) dose C (15,000 nematocysts/ml).
Short
Communications
499
Table 1. (a) Effects of crude extracts of A. aequorea, R. pulmo and A. sulcataon V79 cells, after 3 hr of treatment, evaluated by neutral red assay* and (b) DNA fragmentation in V79 cells after 1 hr exposure to A. aequorea, R. pulmo and A. sulcata crude toxins?
(a) Species Control Aequorea aequorea
R. pulmo
A. sulcata
Dose (N/ml)
No. of samples
Survival % (mean + SD.)
-
4 2 2 2 4 4 4 4 4 4
100 30.43’ 53.49% 78.641 3.25 k 1.53 35.25 f 17.58 68.06 + 13.41 9.04 * 1.19 55.33 + 7.98 85.64 k 0.59
150,000 30,000 15,000 150,000 30,000 15,000 150,000 30,000 15,000
ICW
ICW
(N/ml)
(N/ml)
76.6 x 10’
187.3 X 10’
39.9 X 10’
118.0 x 10’
65.0 x lo3
138.2 x 10’
@I Species Control A. aequorea R. pulmo A. sulcata N-nitroso-N-methylurea
Dose (N/ml) 150,000 30,000 150,000 (0.3 mM)
No. of samples 5 5 5 5 4
% DNA eluted from the filter (mean + S.D.) 8.7 11.3 10.9 9.7 65.2
k * * f k
2.4 3.5 3.9 3.4 38.0
K, - K, (mean + SD.) K, = 0.00706 f 1.32+ 1.27k 1.19 * 11.60 f
0.0021 0.25 0.34 0.19 3.39
*Per cent survival in relation to control (means and S.D. are calculated on four samples). TData are expressed both as percentage of DNA eluted from the filter and as the relative elution rate (KJKJ. The initial rate constant of DNA elution, K (ml - ‘), was calculated from the equation K = ( - InFR)/ V, where FR is the fraction of DNA retained on the filter and V is the eluting volume (13 ml). As a first approximation, K is directly proportional to the amount of DNA fragmentation (Kohn et al., 1976). TPreliminary results obtained from only two samples. N = Nematocyst number.
Presented results show crude toxins produce evident cytotoxic effects and growth inhibition on V79 cells. Fast action was noted using toxins of R. pulmo, whose tissue cytotoxicity (Allavena et al., 1995) and irritating properties are known, and of A. sulcata, whose venom can produce dermatitis on humans (Maretic and Russell, 1982; Vena et al., 1989) and cytotoxicity on cultured cells (Mariottini et al., 1993). A. aequorea, which was never studied from the toxicological point of view, greatly affects cell growth rate; furthermore, dose increasing did not clearly increases cell mortality; the significance of this phenomenon is not clear and needs further studies. Therefore, there are fast-acting toxins (R. pulmo, A. sulcata) which kill cells rapidly, and slow-acting toxins (A. aequorea) which affect mainly growth rate. It has been suggested that both nematocysts and surrounding tissues are responsible of toxicity (Endean and Noble, 1971; Wittle et al., 1974; Mariscal, 1974); as concerns the studied species, also our results show a high toxicity of extracts containing both nematocyst venoms and tissue components. Therefore, in the complex of toxic phenomena caused by stinging, both components can play an important role; it will be a matter of concern to understand the relationships between these fractions and their participation in the global toxicity.
Acknowledgemenrs-This work was partially supported by a research grant from the W.H.O. Regional Office for Europe within the framework of the Long-term Programme of Pollution Monitoring and Research in
500
Short Communications
the Mediterranean Sea (MED POL Phase II), and by funds ‘60%’ M.U.R.S.T. (University and ScientificTechnological Research Ministry of Italy). We thank Professor A.Martelli (Pharmacology Institute, University of Genova), Professor G.Corte (Immunobiology Department, IST Genova), the ‘Gruppo Sportivo Olimpia Sub Spotorno’ and the ‘Cooperativa Pescatori di Camogli’.
REFERENCES Allavena, A., Mariottini, G. L., Martelli, A. and Carli, A. (1995) In uitro cytotoxic activity of the tissular toxin of Rhizostoma vulmo Aeassiz (Cnidaria: Scvnhozoa). Pharmac. Toxic. 76 Suppl. I, 31. Arillo, A., Burlando, B., C&Ii, A: M. and Ma&ttini, 6. L. (1994) Mitochondriai alteration caused by cnidarian toxins: a preliminary study. Boll. Sot. it. Biol. Sper. 70, 307-313. Babich, H. and Borenfreund, E. (1987) Structure-activity relationship (SAR) models established in oitro with the neutral red cytotoxicity assay. Toxicol. In Vitro 1, 3-9. Batista, U. and Jezernik, K. (1992) Morphological changes of V-79 cells after equinatoxin II treatment. Cell Biol. ht. Rep. 16, 115-123. Batista, U., Macek, P. and Sedmak, B. (1986) The influence of equinatoxin II on V-79-379 A cell line. Period Biol. 88, 97-98.
Batista, U., Macek, P. and Sedmak, B. (1990) The cytotoxic and cytolytic activity of equinatoxin II from the sea anemone Actinia equina. Cell Biol. Int. Rep. 14, 1013-1024. Btress, L., Btress, R. and Wunderer, G. (1975) Purification of three polypeptides with neuro- and cardiotoxic activity from the sea anemone Anemonia sulcata. Toxicon 13, 359-367. Bernheimer, A. W. and Avigad, L. S. (1976) Properties of a toxin from the sea anemone Stoichactis helianthus, including specific binding to sphingomyelin. Proc. natn. Acad. Sci. U.S.A. 73, 467471. Brambilla, G., Robbiano, L., Caselli, E., Martelli, A., Turmolini, F. and Mazzei, M. (1988) Cytotoxic, DNA-damaging and mutagenic properties of 2,6-dimethoxy- 1,4benzoquinone, formed by dimethophrine nitrite interaction. J. Pharmac. exp. Ther. 224, 101l-1015. Cariello, L., Romano, G., Spagnuolo. A. and Zanetti, L. (1988) Isolation and partial characterization of rhizolysin a high molecular weight protein with hemolytic activity, from the jellyfish Rhizostoma pulmo. Toxicon 26, 1057-1065.
Carli, A., Mariottini, G. L. and Pane, L. (in press) Ecological and medical aspects of jellyfish poisoning. MAP Technical Reports Series, Athens: UNEP. Endean, R. and Noble, M. (1971) Toxic material from the tentacles of the cubomedusan Chironexjleckeri. Toxicon 9, 255-264.
Giraldi, T., Ferlan, I. and Romeo, D. (1976) Antitumor activity of equinatoxin. Chem. Biol. Interact. 13,199-203. Kohn, K. W., Erickson, L. L., Ewig, R. A. G. and Friedman, C. A. (1976) Fractionation of DNA from mammalian cells by alkaline elution. Biochemistry 15, 46294637. Macek, P. and Lebez, D. (1988) Isolation and characterization of three lethal and hemolytic toxins from the sea-anemone Actinia equina L. Toxicon 26, 441451. Maretic, Z. and Russell, F. E. (1982) Some epidemiological and clinical aspects of stings by the anemone Anemonia sulcata. Toxicon 20, 360-361.
Mariottini, G. L., Bussotti, S. and Carli, A. (1993) Cytotoxic effects produced on a continuous cell line by the nematocyst venom of Anemonia sulcatu (Cnidaria, Anthozoa). I. Cytotechnology 11S, 158-159. Mariscal, R. N. (1974) Nematocysts. In: Coelenterate Biology. Reviews and New Perspectives, pp. 129178 (Muscatine, L. and Lenhoff, H. M., Eds). New York: Academic Press. Mazzei, M., Garzoglio, R., Mariottini, G. L. and Carli, A. (1994) HPLC separation of toxic fraction components extracted from planktonic and benthic Cnidaria. Boll. Sot. it. Biol. Sper. 70, 143-151. Mazzei. M.. Allavena, A.. Garzoelio. R.. Mariottini. G. L. and Carli. A. (1995) Chemical and chromatoaraohic characteristics of toxin from the jellyfish Rhizostoma pulmo Agassiz (Chidaria: Scyphozoa). Pharmac-Tdxic. 76 Suppl. I, 38.. Quadrifoglio, F., Avian, M., Del Negro, P., Princi, T., Scuka, M., Gavinelli, E. and Rottini Sandrini, L. (1986) Nematocisti e tossine di Pelagia noctiluca. Atti XVIII Congr. S.I.B.M., Cesenatico. Nova Thalassia 8 Suppl. 3, 155-162.. Salleo, A., Calabrese, L., Barra, D. and La Spada, G. (1986) Characterization of protein components of the capsule fluid and of the capsule wall of the nematocysts of Pelagia noctiluca. Nova Thalassia 8 Suppl. 2, 119-122. Vena, G. A., Fiordalisi, F. and Angelini, G. (1989) Dermatite da contatto e reazione anafilattoide da Anemonia sulcata. Dermatologia in Posters, Rim-none Interregionale SIDEV Centro-Sud e Isole, Cosenza, November 1988, Cilag S.p.A. Ed. Wittle, L. W., Scura, E. D. and Middlebrook, R. E. (1974) Stinging coral (Millepora tenera) toxin: a comparison of crude extracts with isolated nematocyst extracts. Toxicon 12, 481486.
0041-0101(95)00157-3
0
Toxicon. Vol. 34, No. 4, pp. 496-330, 1996 1996 Elsevier seiclla Ltd. All ri&s reserved Printed in Greal Britain co41~101/% SIS.00 + 0.00
TOXICITY OF JELLYFISH AND SEA-ANEMONE VENOMS ON CULTURED V79 CELLS A. CARLI,‘*
S. BUSSOTTI,’
G. L. MARIOTTINI’
and L. ROBBIANO*
‘Istituto di Scienze Ambientali Marine, Cattedra di Planctologia, Universiti di Genova, Viale Benedetto XV 5, 16132 Genova, Italy and ‘Istituto di Farmacologia, UniversS di Genova, Viale Benedetto XV 2, 16132 Genova, Italy (Received 9 August 1995; accepted 22 November 1995)
A. Carli, S. Bussotti, G. L. Mariottini and L. Robbiano. Toxicity of jellyfish and sea-anemone venoms on cultured V79 cells. Toxicon 34, 496-500, 1996.-Cnidarian toxins exert an influence on human activities and public health. The cytotoxicity of crude toxins (nematocyst and surrounding tissue venom) of Aequorea aequorea, Rhizostoma pulmo and Anemonia sulcata was assessed on V79 cells. Rhizostoma pulmo and Anemonia sulcata crude venoms showed remarkable cytotoxicity and killed all treated cells at highest tested concentration within 2 and 3 hr, respectively. Aequorea aequorea crude venom greatly affected growth rate during long-term experiments. No genotoxic effect was observed. Copyright 0 1996 Elsevier Science Ltd.
Cnidarian toxications are a world-wide health problem and can seriously affect some working (fishery) and tourist (bathing) activities. Some studies concerning Mediterranean Cnidaria led to toxin characterization of Actinia equina (Macek and Lebez, 1988), Anemonia sulcata (BCress et al., 1975), and partly of Pelagia noctiluca (Quadrifoglio et al., 1986; Salleo et al., 1986) and Rhizostoma pulmo (Cariello et al., 1988; Mazzei et al., 1994, 1995). Actually, excluding haemolytic effects, the cell-damaging action of Cnidarian toxins is greatly unknown (Bernheimer and Avigad, 1976; Giraldi et al., 1976; Batista et al., 1986, 1990; Batista and Jezernik, 1992; Mariottini et al., 1993; Allavena et al., 1995). Taking into account that toxins are located both in nematocysts and in surrounding tissues (Mar&al, 1974), the aim of this investigation was to determine the cytotoxicity in vitro of crude extracts obtained from nematocysts and tissues of Aequorea aequorea, Rhizostoma pulmo and Anemonia sulcata. Jelly&h A. aequorea (Hydrozoa) and R. pulmo (Scyphozoa) were collected in the Ligurian Sea and in the Northern Tyrrhenian Sea along the Eastern Sardinian coast, and kept at - 20°C until utilization. Sea-anemones A. sulcata (Anthozoa), were collected along the cliff of Sori (Ligurian Sea) and the coast of Porto Cervo (Northern Tyrrhenian * Author to whom correspondence should be addressed. 496
Short Communications
497
Sea), and were indefinitely maintained in aquaria. Tentacles of A. aequorea and A. sulcata and oral arm parts of R. pulmo were treated as reported (Arillo et al., 1994; Mazzei et al., 1994). Three extract amounts per species were prepared in relation to the counted nematocyst number (N), used as an estimate of toxin rate assuming that most of toxin is stored in nematocysts (dose A: 150,000 N/ml; dose B: 30,000 N/ml; dose C: 15,000 N/ml) and tested on V79 fibroblasts (NIH, Coriell Institute for Medical Research, Camden, NJ) for 1, 2 and 3 hr, and 5 days. Cell viability was evaluated both by Trypan Blue dye exclusion (TB; short- and long-term cytotoxicity) and neutral red assay (NR; according to Babich and Borenfreund, 1987; short-term citotoxicity). Cell morphology, detachment, colony forming efficiency (CFE) and growth rate (number of doublings) were also considered. The DNA fragmentation (single-strand breaks and/or alkali-labile sites) was evaluated by the alkaline elution technique (Brambilla et al., 1988). At the experimental temperature (37°C) no significant pH shifting from normal values was induced in culture medium by crude toxins. After 1 hr treatment with doses A and B of R. pulmo and dose A of A. sulcata cells appeared rounded and devoid of their typical extensions, several of them were detached from culture flasks. The cells treated with other doses of R. pulmo and A. sulcata and with all doses of A. aequorea showed usual morphology. After 2 hr cells also treated with dose B of A. sulcata showed the above reported morphological changes. No attached cells treated with dose A of R. pulmo and A. sulcata were observed after 3 hr, while all doses C and the dose B of A. aequorea did not appear different from control. Figure 1 shows short- and long-term cytotoxicity assessed by TB. The dose A of R. pulmo and A. sulcata induced fast toxic response, produced a noticeable decrease of cell viability after 1 hr and killed all cells after 2 and 3 hr, respectively. For A. aequorea dose A produced high cytotoxicity only after 3 hr. High toxic effects of dose B within the first hour and substantially no further toxicity were noticed by utilizing R. pulmo extracts, while for A. sulcata the effects were evident only after 3 hr. The dose C of the anemone produced slower effects than in R. pulmo. For A. aequorea data show a scarce influence of dose B on cell viability with effectiveness during the third hour, and a similar trend of doses A and C. During long-term experiments also the dose B of A. sulcata killed all cells and the dose C was highly toxic. Doses B and C of R. pulmo showed a remarkable toxicity. The toxin of A. aequorea allowed cell survival also with dose A. Overall, the toxicity of A. aequorea was similar at all doses and highly affected cell growth rate, as showed by calculated doublings; a slow growth rate in comparison with control was observed also using crude extracts of R. pulmo and A. sulcata (Carli et al., in press). NR results after 3 hr of treatment (Table la) are similar to those obtained using TB: lcso and IQ,, showed a strong cytotoxicity of R. pulmo and A. sulcata crude toxins and lower effects of A. aequorea. CFE was evaluated only at doses that permitted cell survival after the long-term experiment; preliminary results showed the dose C of R. pulmo allowed high CFE (75.5%; within normal values for V79 cells); values close to normality were observed also using dose B of R. pulmo (62.5%), doses B (64.0%) and C (65.0%) of A. aequorea, and dose C of A. sulcata (69.5%); on the contrary, dose A of A. aequorea showed remarkable effects (35.0%). Data provided by DNA damage/alkaline elution (Table lb) indicate that 1 hr treatment with subtoxic concentration of nematocysts of the three Cnidarian species did not cause increase of DNA elution rate, that might indicate the occurrence of DNA fragmentation. Under identical experimental conditions, a high frequency of DNA breaks was induced by 1 hr of exposure to 0.3 mM N-nitroso-N-methylurea used as positive control.
498
Short Communications
1 hour
2 hours
1 hour
2 hours
3 hours
100 90
80 0
70
S 2
60
%
50
B
40
E
30 20 10 0 3 hours
5 days
T 100
l
90
-.
80
-.
70
-.
60
..
50
..
40
-.
30
..
20
-.
C
10 -.
To
1 hour
2 hours
3 hours
5 days
timr
Fig. 1. Per cent survival in relation to controls of V79 cells treated with crude toxins extracted from A. aequorea, R. pulmo and A. sulcata during short- (1, 2 and 3 hr) and long-term (5 days) experiments (means and S.D. are calculated on four samples): (a) dose A (150,000 nematocysts/ml); (b) dose B (30,000 nematocysts/ml); (c) dose C (15,000 nematocysts/ml).
Short
Communications
499
Table 1. (a) Effects of crude extracts of A. aequorea, R. pulmo and A. sulcataon V79 cells, after 3 hr of treatment, evaluated by neutral red assay* and (b) DNA fragmentation in V79 cells after 1 hr exposure to A. aequorea, R. pulmo and A. sulcata crude toxins?
(a) Species Control Aequorea aequorea
R. pulmo
A. sulcata
Dose (N/ml)
No. of samples
Survival % (mean + SD.)
-
4 2 2 2 4 4 4 4 4 4
100 30.43’ 53.49% 78.641 3.25 k 1.53 35.25 f 17.58 68.06 + 13.41 9.04 * 1.19 55.33 + 7.98 85.64 k 0.59
150,000 30,000 15,000 150,000 30,000 15,000 150,000 30,000 15,000
ICW
ICW
(N/ml)
(N/ml)
76.6 x 10’
187.3 X 10’
39.9 X 10’
118.0 x 10’
65.0 x lo3
138.2 x 10’
@I Species Control A. aequorea R. pulmo A. sulcata N-nitroso-N-methylurea
Dose (N/ml) 150,000 30,000 150,000 (0.3 mM)
No. of samples 5 5 5 5 4
% DNA eluted from the filter (mean + S.D.) 8.7 11.3 10.9 9.7 65.2
k * * f k
2.4 3.5 3.9 3.4 38.0
K, - K, (mean + SD.) K, = 0.00706 f 1.32+ 1.27k 1.19 * 11.60 f
0.0021 0.25 0.34 0.19 3.39
*Per cent survival in relation to control (means and S.D. are calculated on four samples). TData are expressed both as percentage of DNA eluted from the filter and as the relative elution rate (KJKJ. The initial rate constant of DNA elution, K (ml - ‘), was calculated from the equation K = ( - InFR)/ V, where FR is the fraction of DNA retained on the filter and V is the eluting volume (13 ml). As a first approximation, K is directly proportional to the amount of DNA fragmentation (Kohn et al., 1976). TPreliminary results obtained from only two samples. N = Nematocyst number.
Presented results show crude toxins produce evident cytotoxic effects and growth inhibition on V79 cells. Fast action was noted using toxins of R. pulmo, whose tissue cytotoxicity (Allavena et al., 1995) and irritating properties are known, and of A. sulcata, whose venom can produce dermatitis on humans (Maretic and Russell, 1982; Vena et al., 1989) and cytotoxicity on cultured cells (Mariottini et al., 1993). A. aequorea, which was never studied from the toxicological point of view, greatly affects cell growth rate; furthermore, dose increasing did not clearly increases cell mortality; the significance of this phenomenon is not clear and needs further studies. Therefore, there are fast-acting toxins (R. pulmo, A. sulcata) which kill cells rapidly, and slow-acting toxins (A. aequorea) which affect mainly growth rate. It has been suggested that both nematocysts and surrounding tissues are responsible of toxicity (Endean and Noble, 1971; Wittle et al., 1974; Mariscal, 1974); as concerns the studied species, also our results show a high toxicity of extracts containing both nematocyst venoms and tissue components. Therefore, in the complex of toxic phenomena caused by stinging, both components can play an important role; it will be a matter of concern to understand the relationships between these fractions and their participation in the global toxicity.
Acknowledgemenrs-This work was partially supported by a research grant from the W.H.O. Regional Office for Europe within the framework of the Long-term Programme of Pollution Monitoring and Research in
500
Short Communications
the Mediterranean Sea (MED POL Phase II), and by funds ‘60%’ M.U.R.S.T. (University and ScientificTechnological Research Ministry of Italy). We thank Professor A.Martelli (Pharmacology Institute, University of Genova), Professor G.Corte (Immunobiology Department, IST Genova), the ‘Gruppo Sportivo Olimpia Sub Spotorno’ and the ‘Cooperativa Pescatori di Camogli’.
REFERENCES Allavena, A., Mariottini, G. L., Martelli, A. and Carli, A. (1995) In uitro cytotoxic activity of the tissular toxin of Rhizostoma vulmo Aeassiz (Cnidaria: Scvnhozoa). Pharmac. Toxic. 76 Suppl. I, 31. Arillo, A., Burlando, B., C&Ii, A: M. and Ma&ttini, 6. L. (1994) Mitochondriai alteration caused by cnidarian toxins: a preliminary study. Boll. Sot. it. Biol. Sper. 70, 307-313. Babich, H. and Borenfreund, E. (1987) Structure-activity relationship (SAR) models established in oitro with the neutral red cytotoxicity assay. Toxicol. In Vitro 1, 3-9. Batista, U. and Jezernik, K. (1992) Morphological changes of V-79 cells after equinatoxin II treatment. Cell Biol. ht. Rep. 16, 115-123. Batista, U., Macek, P. and Sedmak, B. (1986) The influence of equinatoxin II on V-79-379 A cell line. Period Biol. 88, 97-98.
Batista, U., Macek, P. and Sedmak, B. (1990) The cytotoxic and cytolytic activity of equinatoxin II from the sea anemone Actinia equina. Cell Biol. Int. Rep. 14, 1013-1024. Btress, L., Btress, R. and Wunderer, G. (1975) Purification of three polypeptides with neuro- and cardiotoxic activity from the sea anemone Anemonia sulcata. Toxicon 13, 359-367. Bernheimer, A. W. and Avigad, L. S. (1976) Properties of a toxin from the sea anemone Stoichactis helianthus, including specific binding to sphingomyelin. Proc. natn. Acad. Sci. U.S.A. 73, 467471. Brambilla, G., Robbiano, L., Caselli, E., Martelli, A., Turmolini, F. and Mazzei, M. (1988) Cytotoxic, DNA-damaging and mutagenic properties of 2,6-dimethoxy- 1,4benzoquinone, formed by dimethophrine nitrite interaction. J. Pharmac. exp. Ther. 224, 101l-1015. Cariello, L., Romano, G., Spagnuolo. A. and Zanetti, L. (1988) Isolation and partial characterization of rhizolysin a high molecular weight protein with hemolytic activity, from the jellyfish Rhizostoma pulmo. Toxicon 26, 1057-1065.
Carli, A., Mariottini, G. L. and Pane, L. (in press) Ecological and medical aspects of jellyfish poisoning. MAP Technical Reports Series, Athens: UNEP. Endean, R. and Noble, M. (1971) Toxic material from the tentacles of the cubomedusan Chironexjleckeri. Toxicon 9, 255-264.
Giraldi, T., Ferlan, I. and Romeo, D. (1976) Antitumor activity of equinatoxin. Chem. Biol. Interact. 13,199-203. Kohn, K. W., Erickson, L. L., Ewig, R. A. G. and Friedman, C. A. (1976) Fractionation of DNA from mammalian cells by alkaline elution. Biochemistry 15, 46294637. Macek, P. and Lebez, D. (1988) Isolation and characterization of three lethal and hemolytic toxins from the sea-anemone Actinia equina L. Toxicon 26, 441451. Maretic, Z. and Russell, F. E. (1982) Some epidemiological and clinical aspects of stings by the anemone Anemonia sulcata. Toxicon 20, 360-361.
Mariottini, G. L., Bussotti, S. and Carli, A. (1993) Cytotoxic effects produced on a continuous cell line by the nematocyst venom of Anemonia sulcatu (Cnidaria, Anthozoa). I. Cytotechnology 11S, 158-159. Mariscal, R. N. (1974) Nematocysts. In: Coelenterate Biology. Reviews and New Perspectives, pp. 129178 (Muscatine, L. and Lenhoff, H. M., Eds). New York: Academic Press. Mazzei, M., Garzoglio, R., Mariottini, G. L. and Carli, A. (1994) HPLC separation of toxic fraction components extracted from planktonic and benthic Cnidaria. Boll. Sot. it. Biol. Sper. 70, 143-151. Mazzei. M.. Allavena, A.. Garzoelio. R.. Mariottini. G. L. and Carli. A. (1995) Chemical and chromatoaraohic characteristics of toxin from the jellyfish Rhizostoma pulmo Agassiz (Chidaria: Scyphozoa). Pharmac-Tdxic. 76 Suppl. I, 38.. Quadrifoglio, F., Avian, M., Del Negro, P., Princi, T., Scuka, M., Gavinelli, E. and Rottini Sandrini, L. (1986) Nematocisti e tossine di Pelagia noctiluca. Atti XVIII Congr. S.I.B.M., Cesenatico. Nova Thalassia 8 Suppl. 3, 155-162.. Salleo, A., Calabrese, L., Barra, D. and La Spada, G. (1986) Characterization of protein components of the capsule fluid and of the capsule wall of the nematocysts of Pelagia noctiluca. Nova Thalassia 8 Suppl. 2, 119-122. Vena, G. A., Fiordalisi, F. and Angelini, G. (1989) Dermatite da contatto e reazione anafilattoide da Anemonia sulcata. Dermatologia in Posters, Rim-none Interregionale SIDEV Centro-Sud e Isole, Cosenza, November 1988, Cilag S.p.A. Ed. Wittle, L. W., Scura, E. D. and Middlebrook, R. E. (1974) Stinging coral (Millepora tenera) toxin: a comparison of crude extracts with isolated nematocyst extracts. Toxicon 12, 481486.