Changes of ICE protease activities caused by toxic supernatants of dinoflagellates (Prorocentrum species) from marine algal blooms

Europ. J. Protistol. 35, 267-274 (1999) October 15, 1999 http://www.urbanfischer.de/journalslejp

European Journal of

PROTISTOLOGY

Changes of ICE Protease Activities Caused by Toxic Supernatants of Dinoflagellates (Prorocentrum Species) from Marine Algal Blooms Sanja Perovic', Christian Wetzler', Franz Brurnrner-, Malte Elbrachter", Laszlo Tretter', Antje Wichels 5 , Werner E.G. MOiler' and Heinz C. Schroder!" 'Institut fur Physiologische Chemie, Abteilung fur Angewandte Molekularbiologie, Universitat Mainz, Duesbergweg 6, D - 55099 Mainz, Germany; Fax: ++49-6131-395243; E-mail: [email protected] 2 Biologisches Institut, Abteilung Zoologie, Universitat Stuttgart, Pfaffenwaldring 57, D - 70569 Stuttgart, Germany 3 Forschungsinstitut Senckenberg, Wattenmeerstation SyltdesAWl, HafenstraBe 43, D - 25992 ListiSylt, Germany 4 Semmelweis University of Medicine, POB 262, H - 1444 Budapest, Hungary 5 Biologische Anstalt Helgoland, Abteilung Meeresmikrobiologie, D - 27483 Helgoland, Germany

Summary Marine phytotoxins may become a major health problem for humans because of their ability to contaminate seafood and to cause shellfish poisoning. In this report, the cytotoxic effects and the effects on intracellular caspase activities of culture supernatants from different dinoflagellate Prorocentrum clones were determined. Among the clones tested, P. tepsium BAH ME-140 and P. lima BAH ME-130 Kl and K2 clones but not P. minimum and P. micans were found to be toxic on rat pheochromocytoma PC12 cells, mouse lymphoma L5178Ycells and rat primary neurons. A significant increase in the specificactivities of caspase 1 (ICE), caspase 3 (CPP32) and caspase 6 (Mch2) to 149-167% was observed after treatment of neurons with P. lima BAH ME-130 K2 supernatant for 72 h; in PC12 cells, the increase in these enzyme activities was much smaller. An even stronger and faster effect on caspase activities, compared to the K2 clone, was observed following treatment of PC12 cells and neuronal cells with P. lima BAH ME-130 Kl supernatant. The maximal increase in caspase activities in PC12 cells (CPP32, 364%; and Mch2, 166%) and in neurons (CPP32, 162%; and Mch2, 111%) was observed after 24 h; no significant change of ICE activity was found during that incubation period. After 48 h the specific activities of all caspases strongly decreased. Incubation of PC12 cells with P. tepsium BAH ME140for 24 h had almost no effect on caspase activities, while a small increase in CPP32- (148%) and Mch2- (115%) but not in ICE-activity was detected after 48 h. In neurons, only an increase in CPP32 activity (to 130%) was observed with this dinoflagellate supernatant after 24 h. The P. lima protein phosphatase inhibitor okadaic acid (0.5 ng/ml) *corresponding author © 1999 by Urban & FischerVerlag

caused a time-dependent increase in caspase activities in PC12 cells. A much higher effect was observed in neuronal cells; after 72 h, the specific activities of ICE, CPP32 and Mch2 increased to 295%, 146% and 235%, respectively. These results indicate that disturbances of caspase activities may contribute to the neurotoxic effects of certain dinoflagellate supernatants. Key words: Marine toxins; Algal blooms; Caspases; ICE; CPP32; Mch2; Cell viability; Dinoflagellate; Prorocentrum.

Introduction Toxins produced by marine phytoplankton are a potential risk of human poisoning, resulting in serious disorders [6,34]. Paralytic and diarrhoetic shellfish poisoning in man is mainly caused by the ingestion of shellfish meat (clams, mussels, and oysters) that have accumulated toxins from certain dinoflagellates [23, 48]. Natural or anthropogenic blooms ("red tides") of toxic dinoflagellates, e.g. Alexandrium tamarense or Prorocentrum lima, in coastal waters have been reported throughout the world [1, 16,24,36,40,45]. Sporadic outbreaks of paralytic shellfish poisoning has also become a major problem in areas with intense mariculture [2]. However, there is little knowledge about the causative organisms and the mechanisms of their toxic effects. Several marine dinoflagellate and diatom species are capable of toxin synthesis [28, 39]. A number of para0932-4739/99/35/03-267 $ 12.00/0

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lytic and diarrhoetic shellfish poisoning toxins has been isolated from these organisms and characterized [44]. The unicellular marine dinoflagellate, Prorocentrum lima, has been recognized as a producer of the tumour promoter okadaic acid. Diarrhoetic shellfish poisoning in Europe is mainly due to this toxin [35], but other dinoflagellate toxins that have been identified in crude extracts from P. lima, such as dinophysistoxins 1, 2 and 4 (DTX-l, 2 and 4), may be important, too [11, 20,30]. The effects of okadaic acid have been studied in rat uterus [4], rat myometrium [10], human bronchus [31] and mammalian fibroblast cell lines [15]. Okadaic acid was found to induce a dose-dependent contraction of the isolated uterus and a series of contractions and relaxations of the isolated bronchus. In calcium-free medium the contractile effects of okadaic acid were reduced [4, 10,31]. The harmful effects of dinoflagellate toxins may be caused by induction of the apoptotic pathway. Apoptosis is characterized by a series of distinct morphological and biochemical changes. One of the best described pro-apoptotic genes, ced-3, encodes a Caenorhabditis elegans protein that is highly homologous to mammalian interleukin-l converting enzyme (ICE). ICE is the first identified member of a new class of cystein proteases. It is expressed -like all other caspases - as a precursor that must be proteolytically processed to become an active enzyme [51]. CPP32, an ICE-like cysteine protease, has also been implicated in the pathway of apoptosis in mammalian cells. When activated, CPP32 specifically cleaves poly(ADP-ribose) polymerase and sterol regulatory element binding proteins [26,49]. The poly(ADP-ribose) polymerase is assumed to be involved in DNA repair, and genome surveillance and integrity [13]. Another caspase participating in the cell death pathway, Mch-2, has been shown to be located at the same chromosome as CPP32 [32]. In this paper, we investigated, in PC12 cells and rat primary neuronal cells, the effects of different dinoflagellate (Prorocentrum spp) supernatants on activities of certain caspases (ICE, CPP32, and Mch2) involved in apoptosis. The changes in caspase activities induced by the dinoflagellate supernatants were compared with the toxicity of these supernatants.

Materials and Methods Materials: Okadaic acid, 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MIT), poly-i.-Iysine (M, > 300,000), insulin, progesterone, putrescine, holo-transferrin (bovine), horse serum (HS), RPMI 1640medium, Dulbecco's modified Eagle's medium/1.0 giL glucose(DMEM) and Dulbecco's modified Eagle's medium/4.5 giL glucose (DMEM/

HG) were obtained from Sigma (Deisenhofen, Germany); Hank's balanced salt solution without CaZ+ andMgz+ (HBSS), t-glutamine and trypsin were from Biochrom (Berlin, Germany); fetal calf serum (FCS) was from Gibco (Berlin, Germany); bovine serum albumin (BSA) from Roth (Karlsruhe, Germany); and mouse anti-neurofilament 68 kDa antibody and mouse anti-glial fibrillary acidic protein (GFAP) antibody from Roche Diagnostics GmbH (Mannheim, Germany). Isolation and cultivation of dinoflagellates: The dinoflagellates were grown in f/2 medium [17]in non-axenic batch cultures at a temperature of 17 ± 1°C under 12:12 light/dark cycle and exposed to 25 pE . m-z . S-I. The supernatants were obtained during the exponential growth phase. Prior to use, all supernatants were filtrated through 0.2 pm filters. Cells: Rat cortical cell cultures were prepared from the brains of 18-19 days old Wistar rat embryos as described elsewhere [37]. Briefly,isolated cerebral hemispheres were placed into Caz+ - and MgZ+-free HBSS. After dissociation of the brain tissue in HBSSusing 0.025% trypsin (10 min; 37°C; reaction stop by addition of 10% FCS) and centrifugation, the pelleted neuronal cells were resuspended in DMEM/HG, containing 2 mM r.-glutamine, 100 mUlL of insulin and 10% FCS. The cells were seeded into poly-L-Iysine (5 pg/rnl, 300 pl/cm') coated plastic dishes at 2.0xl0 s cells/em'. Two days later, DMEM/HG/I0% FCS was removed and the cells were further cultivated in DMEM/HG serum-free medium supplemented with 0.1% BSA,2 mM L-glutamine, 100pg/rnl of transferrin, 100 mUlL of insulin, 16 pg/rnl of putrescine, 6.3 ng/ml of progesterone, and 5.2 ng/ml of sodium selenite (NazSeO}). The cultures were analyzed seven days after isolation by immunostaining using anti-neurofilament 68 kDa as marker for neurons and anti-GFAP as marker for glialcells. The percentage of neurons amounted to > 80%; the other cells were GFAP-positive. PC12 cells were grown in DMEM/I0% FCS/5% HS. The cellswere passagedtwice per week at a 1:10 ratio. L5178Y cells were maintained in RPMI 1640/10% FCS. The cellswere subcultured twice per week at a 1:160ratio. All cells were kept in an atmosphere of 95% air and 5% COz at37C. Treatment of cellsfor the determination of cell viability • PC12 cells: PC12 cells were seeded at 1.4xl0 4 cells/em? and maintained in DMEM/I0% FCS/5% HS. 24 hours later dinoflagellate supernatants were added to the cultures. The TC so values were determined after incubation of the cells for 72 h in the presence of different amounts of dinoflagellatesupernatants; the final volume was 200 ul. Control cultures contained an adequate volume of f/2 medium instead of dinoflagellate supernatant. Following incubation, the cell viability was determined using MTT assay. • L5178Y cells: The cells were seeded at 8xl0} cells/ml of RPMI 1640/10% FCS. Dinoflagellate supernatants were added to the cells immediately after seeding; the final volume was 200 pl. In the controls, the cells were incubated with an adequate volume of f/2 medium. The MTT assay was performed after an incubation period of 72 h. • Neurons: Two days after isolation serum-containing medium was removed and exchanged with DMEM/HG serum-free medium. The neuronal cultures (final assay volume, 200 pl) were treated with dinoflagellate supernatants to

Effect of Dinoflagellate Toxins on ICE Proteases

estimate the TC so value; f/2 medium was used as a control. 72 hours later the cell viability was determined. MTT assay: The cell viability was analyzed using the MTT assay [43]. The evaluation was performed in 96-well plates at 595 nm using an ELISA plate reader (BioRad 3550, equipped with the program NCIMR II1B). The results are expressed as TC so values representing the numbers of dinoflagellate cells per one pl of dinoflagellate supernatant which cause 50% cell death after addition to 100 pl of MTT assay. The viability of the control assays (addition of f/2 medium alone) was set at 100%. Monitoring of caspase activity: In all sets of experiments PC12 (1.4-2.4xl04 cells/em') and rat primary neuronal cells (1.7xl0 s cells/em') were incubated with different concentrations of dinoflagellate supernatants. For measurements of caspases the TC so value was used (Table 1). In one set of experiments the cells were incubated with 0.5 ng/ml of okadaic acid. The activities of caspases 1 (ICE), 3 (CPP32) and 6 (Mch2) were determined 24, 48 and 72 hours after addition of the dinoflagellate supernatants. In brief, after incubation (at 37°C) the cells were collected by centrifugation (150xg, 6 min), washed twice in lxPBS pH 7.4, and lysed in 1 mllysis buffer (10 mM HEPES-KOH, 5 mM dithiothreitol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 0.1% (w/v) 3-[(3cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS); pH 6.5) for 20 min on ice. For determination of caspases the supernatant, after centrifugation at 12,000xg for 15 min, was used. The volume of the final reaction mixture was 250 pl; 100 pl of supernatant was mixed with 150 pl of 2xICE buffer (40 mM HEPES-KOH, 1 mM phenylmethylsulfonyl fluoride, 4 mM dithiothreitol and 20% (v/v) glycerol; pH 7.2) supplemented with 12 mM 4-methy1coumaryl7-amide (MCA) substrate. For determination of caspase-l (ICE), Ac-Tyr-Val-Ala-Asp-MCA was used as substrate; for caspase-3, Ac-Asp-Glu-Val-Asp-MCA substrate; and for caspase-6, Ac-Val-Glu-Ile-Asp-MCA substrate (Peptide Institute, Minoh-shi, Osaka, Japan). As a control 100 pl lysis buffer plus 150 pl of 2xICE buffer/MCA substrate was used. The enzymatic reaction was measured (excitation 355 nm; emission 460 nm) every 60 min during 4 hours using Fluoroskan (Labsystems Fluoroskan II; equipped with the program EIA; Version 2.0; Flow Laboratories, 1989). The enzyme activities were measured as OD 46013 h per pg of protein and are given as percentage of control (set at 100%). The protein concentration was determined by Lowry method [27]. Statistics: The results were analyzed by paired Student's t-test [41].

Results Effect of dinoflagellate supernatants on viability of PC 12 and L5178Y cells The TC so values for Prorocentrum tepsium BAH ME-140 [Elbrachter et aI., in preparation] were found to be 115.5 cells/pl in both PC12 and L5178Y cells after an incubation period of 72 h (Table 1). Both the P. lima BAH ME-130 Kl and K2 clones showed different toxicity on the cells. The cytotoxic effect of the P. lima BAH ME-130 K2 supernatant was 2-fold lower in

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Table 1. Effect of supernatants from different dinoflagellate Prorocentrum clones on viability of PC12 cells, L5178Y cells and primary neuronal cells. Cells were incubated with Prorocentrum supernatants as described under Material and Methods. The MTT assay was performed after an incubation period of 72 h. The results are expressed as TC so value (based on 1 pl of dinoflagellate supernatant per 100 pl of assay); n = 16; n.t., not toxic. Prorocentrum clone

P. minimum Kl P. minimum K2 P.lima BAH ME-130 Kl P. lima BAH ME-130 K2 P. micans BAH ME-004 Kl P. micans BAH ME-004 K2 P. tepsium BAH ME-140

PC12 cells (cells/ul) n.t n.t. 42.9 49.5

L5178Y cells (cells/pl) n.t.

Neurons (cells/pl)

8.6 99.0

n.t. n.t. 1.5 24.8

n.t, n.t,

n.t, n.t,

n.t, n.t,

115.5

115.5

3.9

n.t,

L5178Y cells (TC so 99.0 cells/ul) than in PC12 cells (TC so 49.5 cells/pl), while the cytotoxic effect of the P. lima BAH ME-130 Kl supernatant was 5-fold higher in L5178Y cells (TC so 8.6 cells/pl) than in PC12 cells 42.9 cells/pl; Table 1). The P. minimum and P. micans clones were not toxic for PC12 and L5178Y cells under the conditions used (Table 1).

rrc.,

Effect of dinoflagellate supernatants on viability of primary neuronal cells Prorocentrum lima BAH ME-130 Kl and K2, and P. tepsium BAH ME-140 were found to be very toxic for neuronal cells under the conditions used. The TC so values amounted to 3.9 cells/pl for P. tepsium BAH ME-140, 1.5 cells/ul for P. lima BAH ME-130 Kl, and 24.8 cells/ul for the P. lima BAH ME-130 K2 clone (Table 1). All other Prorocentrum clones were not toxic.

Influence of Prorocemrum supernatants on caspase activities in PC 12 cells Caspase activities were examined by measuring the cleavage of the specific fluorogenic peptide substrates, Ac-Tyr-Val-Ala-Asp-MCA (for determination of caspase-l, ICE), Ac-Asp-Glu-Val-Asp-MCA (caspase-3, CPP32), and Ac-Val-Glu-Ile-Asp-MCA (caspase-6, Mch2). Addition of 90 ul/ml of growing dinoflagellate medium (f/2 medium) had almost no effect on the specific activities of ICE, CPP32 and Mch2 in PC12 cells (data not shown). A strong effect on caspase activities was observed after treatment of PC12 cells with the TC so of the P. lima BAH ME-130 Kl supernatant (Fig. 1). In the pres-

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ence of this dinoflagellate supernatant the maximal effect on CPP32 and Mch2 activity was observed after 24 h (Fig. 1). In the case of CPP32 the increase was 364% (p s 0.001) and in the case of Mch2 166% (p ::; 0.001) compared to the control values. Parallelly, ICE activity was reduced to 88% (Fig. 1). After 48 h a dras-

tical decrease in the activities of all caspases was measured; the lowest caspase activities were observed after 72 h. The values were reduced to 43% (ICE), to 34% (CPP32) , and to 24% (Mch2) (p::; 0.001). In contrast, only a small, non-significant (p > 0.05) increase (to 113% after 3 days) in CPP32 activity was

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Fig. 1. Changes of caspases 1 (ICE), 3 (CPP32) and 6 (Mch2) activities in PC12 cells after addition of dinoflagellate supernatants. The caspase activities were determined 24 (open bars), 48 (hatched bars) and 72 h (filled bars) after appl ication of dinoflagellate supernatants (TC so volume) from Prorocentrum lima BAH ME-130 K1 and K2 clone and P. tepsium BAH ME-140. The results are expressed as % of control (set to 100%). The specific enz yme activities of the controls were as follows (given in OD 4w13 h per pg of protein). ICE, 0.83 ± 0.03; CPP32, 2.11 ± 0.34; Mchl, 7.43 ± 0.46 (n = 6; ~'p :s; 0.001).

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Fig. 2. Changes of caspase activities in primary neuronal cells after addition of dinoflagellate supernatants. The activities of ICE, CPP32 and Mchl were determined 24 (open bars), 48 (hatched bars) and 72 h (filled bars) after application of dinoflagellate supernatants (TC so volume) from Prorocentrum lima BAH ME-130 K1 and K2 clone and P. tepsium BAH ME -140. The results are expressed as % of cont rol (set to 100 % ). The sp ecific enz yme activities of the controls were as follows (given in OD 4w13 h per pg of protein). ICE, 0.75 ± 0.14; CPP32, 1.74 ± 0.13; Mch2, 6.05 ± 1.01 (n = 8; "p s. 0.001).

Effect of Dinoflagellate Toxins on ICE Proteases

observed after stimulation of the cells with the TC so of the P. lima BAH ME-DO K2 supernatant (Fig. 1). The ICE and Mch2 activities were almost constant during the.first 48 h after addition of this dinoflagellate supernatant. After 72 h, ICE activity increased to 114% (Fig. 1) and Mch2 activity to 113% . . Incubation of PC12 cells with the TC so of the P. tepSlum BAH ME-140 supernatant resulted only in a small increase (17% compared to the control value) in CPP32 activity after 1 day (Fig. 1). In comparison, almost no change in Mch2 activity (98%) and only a slightly reduced (92%) ICE activity were detected. After 2 days of incubation, the maximal increase in CPP32 and Mch2 activity was measured. The CPP32 activity increased to 148% (p ~ 0.001) and the Mch2 activity to 115%. The ICE activity was still reduced (87%). At the end of the incubation period (3 days) with the P. tepsium BAH ME-140 supernatant the activities of all proteases tested were significantly (p ~ 0.001) reduced to 39% (ICE), 44% (CPP32), and to 35% (Mch2) (Fig. 1).

Influence of dinoflaqellate supernatants on caspase actrvities In neurons In neuronal cells, f/2 medium induced only a small change of caspase activities, like in PC12 cells (data not shown) . Stimulation of the neurons with the TCso of the P. lima BAH ME-DO K2 supernatant resulted in a significant increase in protease activities, after an incubation period of 3 days, to 167% for ICE, to 149% for CPP32 , and to 156% for Mch2 (p ~ 0.001) compared to the controllevels (Fig. 2). The P. lima BAH ME-DO K1 clone was also found to be toxic for the neuronal cells (Table 1) but the timedependence of the changes in caspase activities was different from that for the K2 clone (Fig. 2). The maximal increase in caspase activities in the presence of the TC so of the supernatant of the P. lima BAH ME-DO K1 clone was detected after 1 day [ICE, 102%; CPP32, 162% (p s 0.001), and Mch2, 111%] . After 2 days of incubation all caspase activities tested continuously decreased reaching lowest levels at day 3 (ICE, 77%; CPP32, 59%; and Mch2, 74% compared to control). The increase in caspase activities after treatment of neurons with the TC so of the P. tepsium BAH ME-140 supernatant was high only in the case of CPP32; the ICE activity increased to 106%, the CPP32 activity to 130%, and the Mch2 activity to 105% (Fig. 2). This increase in caspase activities was visible only after 1 day of incubation. After 2 and 3 days the protease activities were lower than in control but the changes observed were not statistically significant. The values were reduced to around 90%.

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Influence of okadaic acid on caspase activities in PC 12 cells and neurons In the presence of 0.5 ng/ml of okadaic acid, there was a slow but non-significant (p > 0.05) time-dependent increase in the specific activities of all three caspases to 105-112% in PC12 cells after 48 h and to 118-123% after 72 h (Fig. 3A). The effect of okadaic acid on the specific activities of ICE, CPP32 and Mch2 in primary neuronal cells was much higher compared to PC12 cells. After 48 h in the presence of 0.5 ng/ml of okadaic acid the specific activity of ICE increased to 124%, the CPP32 activity to 114%, and the Mch2 activity to 138%. 72 h after addition of the drug the caspase activities further increased to 295% (ICE; P s 0.001), 146% (CPP32; P ~ 0.001), and to 235% (Mch2;p s 0.001) (Fig. 3B). An unexpected decrease in the specific activities of the three caspases to 58-71 % compared to the control values was measured after 24 h (Fig. 3B).

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Fig.3. Changes of caspase activities in (A) PC12 cells and (B) rat primary neuronal cells during treatment with okadaic acid. The activities of ICE, CPP32 and Mch2 were determined 24 (open bars), 48 (hatched bars) and 72 h (filled bars) after addition of 0.5 ng/ml of okadaic acid. The results are expressed as % of control (set to 100 % ). For PC12 cells, the specific enzyme activities of the controls are given in Fig. 1; the specific activities of the controls for neurons were as follows (given in OD46013 h per rg of protein) . ICE, 0.82 ± 0.16; CPP32, 2.29 ± 0.18; Mch2, 7.17 ± 0.44 (n = 6; "P s 0.001).

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Discussion In this report, we show that culture supernatants from certain dinoflagellate Prorocentrum clones display a significant cytotoxic effect on rat PC12 cells, mouse lymphoma L5178Y cells and rat primary neurons. Only the supernatants from three clones, P. tepsium BAH ME-140, and P. lima BAH ME-130 Kl and K2 were found to be toxic for all cells tested. These results are in line with previous data obtained by HPLC [21]. Now our results revealed that the dinoflagellate supernatants induce an increase in intracellular caspase activities in PC12 cells and primary neuronal cells. Caspases play a key role in the induction of the apoptotic process [47]. They are homologues of the product of the nematode cell death gene, ced-3. All known members of the ICE/ced-3 family are synthesized as inactive precursors and are proteolytically processed to active enzymes [22]. Caspases are responsible for both the cleavage of lamin Bland the disintegration of the nucleus during apoptosis [3]. Activation of these proteases also results in cleavageof the DNA repair enzyme poly(ADP-ribose) polymerase (pARP) early during apoptosis [25]; this enzyme is specifically cleaved by CPP32 [46]. In addition, proteases are involved in exposure of the inner plasma membrane leaflet lipid, phosphatidylserine, to the outer plasma membrane leaflet during apoptosis [29]. Caspases are sequentially activated during apoptosis [14].The first protein which is cleaved during apoptosis is fodrin. PARP, Ul-70kDa and DNA-PKcs are cleaved by CPP32-like proteases during the second phase of the protease cascade. The third phase involves cleavage of lamin Bl [42]. Treatment with the P. lima BAH ME-130 Kl supernatant resulted in a strong increase in the activities of CPP32 and Mch2 (caspase 3 and 6) in PC12 cells and in the activity of CPP32 in primary neuronal cells already after an incubation period of 24 h, while the activity of ICE was either slightly reduced (PC12 cells) or remained essentially unchangedj neurons). A significant increase in caspase activities (ICE, CPP32, and Mch2) was also observed in neurons treated with the P. lima BAH ME-130 K2 supernatant, but only after an incubation period of 72 h. A prolonged incubation (48 to 72 h) of PC12 cells and neurons in the presence of the P. lima BAH ME-130 Kl supernatant resulted in a decrease in the activities of all three caspases. Treatment of PC12 cells with the supernatant of the P. tepsium BAH ME-140 clone for 24 h caused a moderate increase in CPP32 and Mch2 activity but not in ICE activity after 48 h, while only small changes in caspase activities were observed after 24 h and a significant reduction of caspase activities after 72 h. In neurons, this clone caused only a significant increase in CPP32 activity after 24 h;

lower protease activities were observed after 48 and 72 h. From our results we conclude that the neurotoxic effects of certain dinoflagellate P. lima supernatants are at least partially due to disturbances of intracellular caspase activities. Caspase activation at least by P. lima BAH ME-130 Kl was a very early event (24 h) and there are no hints that caspase activation as a late secondary reaction due to ongoing necrosis had a major influence on the data. Therefore, caspase activation apparently does not reflect a late secondary reaction caused by ongoing necrotic cell death. Functional studies using a broad spectrum caspase inhibitor may give further insight in later processes. P. lima, which was found to be toxic for PC12 cells, L5178Y cells and rat primary neuronal cells, is a known producer of the diarrhoetic shellfish poisoning toxin okadaic acid [8]. Further toxins present in this dinoflagellate are dinophysistoxins 1, 2 and 4 (DTX -1, 2 and 4) [11,20,30]. Okadaic acid, which was found to be capable of increasing caspase activities (this paper), and dinophysistoxin-I and 4 are potent phosphatase inhibitors [20]. Okadaic acid may therefore interfere also with intracellular signalling mechanisms within P. lima. Both a protein kinase A and a type-I protein phosphatase activity, which may be involved in regulation of protein kinase A, have been identified in this dinoflagellate [7, 12]. Okadaic acid and dinophysistoxin-I have also been reported to affect the growth of microalgae; P. lima itself was not affected [50]. Previously we showed that the neurotoxic effects of certain dinoflagellate supernatants (Alexandrium spp) are at least partially caused by disturbances in synaptosomal intracellular calcium levels ([Ca 2+l) [38]. Measurements of [Ca2+1 revealed that the supernatants from the three Prorocentrum clones which were toxic for all cells/cell lines tested, P. tepsium BAH ME-140, and P. lima BAH ME-130 Kl and K2, did not induce calcium influx in both PC12 cells and rat primary neuron al cells, as well as in guinea-pig synaptosomes (unpublished results). Interestingly, the response of rat myometrium to okadaic acid has been shown to depend on calcium, but does not seem to involve calcium entry through dih ydropyridine-sensitive Ca 2+channels [4]. There is a worldwide increase in the frequency of blooms of harmful marine dinoflagellates and microalgae. This may have impacts on both human health [6, 34] and on marine fish populations [9]. Changes in N:P ratios in coastal waters caused by increased nutrient levels due to sewage and polluted water from rivers may be responsible for increased red tide blooms [19]. Phytoplankton growth may be also affected by dissolved organic and inorganic substances produced by fish farming [5]. Moreover, toxic dinoflagellates may be distributed via ballast water of ships [18]. Possible

Effect of Dinoflagellate Toxins on ICE Proteases

strategies to prevent harmful algae blooms may include the control of phosphate pollution of marine environments, e.g. by use of polyphosphate-accumulating bacteria for enhanced biological removal of phosphate from wastewater [33]. In addition, to understand the enzymatic mechanisms and the regulation of the genes involved in the biosynthesis of algal toxins is an important task for future research. Acknowledgements: This work was supported by a grant from the Bundesministerium fur Bildung und Technologie (BMBF Verbundprojekt "TEPS").

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