The Marine Toxin Dinophysistoxin-2 Induces Differential Apoptotic Death of Rat Cerebellar Neurons and Astrocytes

Anabel Pérez-Gómez*, Armando García-Rodríguez*, Kevin J. James{dagger}, Amaia Ferrero-Gutierrez*, Antonello Novelli*,{ddagger},1 and M. Teresa Fernández-Sánchez*,1

* Biochemistry and Molecular Biology Department, University of Oviedo, Oviedo, Spain; {dagger} PROTEOBIO, Mass Spectrometry Center for Proteomics and Biotoxin Research, Department of Chemistry, Cork Institute of Technology, Bishopstown, Cork, Ireland; and {ddagger} Psychology Department, University of Oviedo, Oviedo, Spain

Received February 11, 2004; accepted March 26, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Diarrhetic shellfish poisoning (DSP) toxins of algal origin are frequent contaminants of coastal waters and seafood. The potential risk for human health due to the continuous presence of these toxins in food has not been clearly established. We have used cerebellar primary cultures to investigate the effects of the DSP toxin dinophysistoxin-2 (DTX-2) on central nervous system neurons and glial cells. Exposure to DTX-2 produced neurotoxicity at concentrations starting at 2.5 nM, characterized first by disintegration of neurites and later by cell death. DTX-2-induced neurodegeneration required long exposures (at least 20 h), involved DNA fragmentation and condensation and fragmentation of chromatin, typical hallmarks of apoptosis, and required the synthesis of new proteins. The concentration that reduced by 50% the maximum neuronal survival after 24 h exposure to DTX-2 (EC5024) was ~8 nM. Morphology and viability of glial cells remained unaffected up to at least 15 nM DTX-2. Higher concentrations of the toxin caused strong shrinkage of glial cell bodies and retraction of processes, and a significant reduction of glial cell viability. Glial toxicity by DTX-2 involved typical apoptotic condensation and fragmentation of chromatin. Compared to neurons, the effect on glial cells was a much shorter process, and extensive glial degeneration and death occurred after 7 h exposure to DTX-2 (EC507 ~50 nM; EC5024 ~30 nM). Although further experiments are needed to confirm these toxic actions in vivo, our in vitro data suggest that chronic exposure to amounts of DSP toxins below the current safety regulatory limits may represent a risk for human health that should be taken into consideration.

Key Words: dinophysistoxins; diarrhetic shellfish poisoning; neurotoxicity; apoptosis; cerebellar neurons; glial cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Marine toxins of algal origin are frequent contaminants of coastal waters. These toxins accumulate in the digestive glands of mollusks and marine sponges by filter feeding, and may lead to human intoxication following ingestion of contaminated seafood.

Diarrhetic shellfish poisoning (DSP) is a human gastrointestinal illness caused by the consumption of shellfish contaminated with polyether toxins produced by dinoflagellates belonging to the genera Dinophysis, though some DSP toxins are also produced by benthic species of Prorocentrum (Yasumoto et al., 1979Go, 1984Go). Okadaic acid, its isomer dinophysistoxin-2 (DTX-2), and their methyl homologue dinophysistoxin-1 (DTX-1), are the main DSP toxins. Although okadaic acid is usually the principal toxin in Europe, reports concerning the presence of dinophysistoxins in coastal waters have significantly increased in the last years suggesting the spread of these toxins worldwide. DTX-1 is the dominant toxin in Japan (Yasumoto et al., 1979Go), in Canada (Quilliam et al., 1993Go), and in certain Norwegian fjords (Lee et al., 1989Go), while DTX-2 has been shown to be the predominant toxin in Irish mussels (Carmody et al., 1996Go; McMahon et al., 1996Go). High amounts of DTX-2 have been also detected in shellfish from the Galician region of Spain (Blanco et al., 1995Go) and in Portuguese mussels (Vale and Sampayo, 1996Go, 2000Go). Also, a monitoring program carried out in 1996 and 1997 confirmed for the first time the occurrence of DTX-2 in Adriatic mussels (Pavela-Vrancic et al., 2002Go).

The widespread distribution of DSP toxins in seafood has caused increased concern due to the threat of public health, and has underlined the need for toxicological studies in order to evaluate the potential risk for human health due to the presence of these toxins in food. Up to now most studies have been focused on okadaic acid, while studies regarding dinophysistoxins have been very limited and very few data exist about the action of these compounds on cell survival and physiology. As for DTX-2, toxicological studies have been particularly hampered by the lack of purified material. DTX-2 is produced by phytoplankton belonging to Dinophysis sp., which cannot be maintained in culture, and therefore isolation from wild marine biological materials represents the only source of this toxin.

Cerebellar neurons in primary culture represent an experimental system that has proved very useful in a variety of toxicological studies. These cultures have been extensively used in the study of the biochemical events coupled to neurotoxicity by excitatory amino acids and the conditions controlling excitotoxicity (Fernández-Sánchez and Novelli, 1993Go; Lipsky et al., 2001Go; Novelli et al., 1987Go, 1988Go). Cultured cerebellar neurons have been also identified as a very convenient model for the study of neuronal apoptosis in vitro (D'Mello et al., 1993Go) and extensively used for that purpose thereafter. Moreover, we have found cultured cerebellar neurons to be very useful to investigate the action of different types of marine toxins including okadaic acid (Fernández et al., 1991Go, 1993Go; Fernández-Sánchez et al., 1996Go; Novelli et al., 1992Go). In this study we have used primary cultures of cerebellar neurons to investigate the actions of DTX-2, isolated from contaminated Irish mussels. We also used co-cultures of cerebellar neurons and astrocytes and highly enriched astroglial cultures in order to identify possible differences between the actions of this toxin on neuronal and non-neuronal cells. Our study provides the first data about the mechanisms involved in the toxic effects of DTX-2 on live cells. In view of the high sensitivity of cultured cerebellar neurons to DTX-2 and okadaic acid, this tissue culture system appears to be an appropriate model for the assessment of the potential risk for human health of DSP toxins and for the establishment of safety levels of these compounds in seafood.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals. DTX-2 was isolated from contaminated mussels by Dr. K. James (James et al., 1999Go). Carboxy 2', 7'-dichlorodihydrofluoresceine diacetate (carboxy-H2DCFDA, C-400) was from Molecular Probes (Eugene, OR). All other drugs were from Sigma (St. Louis, MO).

Cell culture. Primary cultures of rat cerebellar neurons were prepared as previously described (Novelli et al., 1988Go). Cytosine arabinoside (10 µM) was added after 20–24 h of culture to inhibit the replication of non-neuronal cells. After 8 days in vitro, morphologically identifiable granule cells accounted for more than 95% of the neuronal population, the remaining 5% being essentially GABAergic neurons. Astrocytes did not exceed 3% of the overall number of cells in culture. Cerebellar neurons were kept alive for more than 40 days in culture by replenishing the growth medium with glucose every four days and compensating for lost amounts of water, due to evaporation. Mixed cerebellar cultures containing neurons and astrocytes and highly enriched astroglial cultures were prepared as described (Suárez-Fernández et al., 1999Go). The presence of microglia in these cultures was determined by OX-42 immunostaining and did not exceed 2% of total cell population in both pure and mixed cultures, and 5% in astroglial cultures. The animal procedures used were in accordance with the protocols approved by the Institutional Animal Care and Use Committee of the University of Oviedo

Neurotoxicology. Neurons were used between 14–20 days in culture. Drugs were added into the growth medium at the indicated concentrations, and neuronal cultures were observed for signs of neurotoxicity thereafter by phase contrast microscopy. To quantify neuronal survival cultures were stained with fluorescein diacetate and ethidium bromide (Fernández et al., 1991Go; Novelli et al., 1988Go), photographs of three randomly selected culture fields were taken and live and dead neurons were counted. Results were expressed as percentage of live neurons. Total number of neurons per dish was calculated considering the ratio between the area of the dish and the area of the pictures (~3000).

Confocal microscopy. Oxygen radical formation was detected with carboxy-2'7'-dichlorodihydrofluoresceine diacetate (carboxy-H2DCFDA). Following uptake, the carboxy-H2DCFDA is converted by endogenous esterases to carboxy-H2DCF, which upon exposure to oxidative species is oxidized to the fluorescent probe carboxy-DCF. Cultures were treated with DTX-2 (10–50 nM) for 6–18 h and then loaded with 20 µM carboxy-H2DCFDA in the culture medium for 1 h. Then, the dye was removed and cultures were washed twice with a buffer containing (in mM): 154 NaCl, 5.6 KCl, 5.6 glucose, 8.6 HEPES, 1 MgCl2, 2.3 CaCl2 (pH 7.4). Carboxy-DCF fluorescence was recorded in a Bio-Rad confocal microscope with a krypton-argon laser excitation source (488 nm). Signals were digitized using Bio-Rad interface and fluorescence intensity was quantified in 10–15 cell bodies per field using the software NIH Image (version 1.61).

DNA fragmentation analysis. Cells were lysed in 10 mM Tris-HCl, 0.5% Triton X-100, 20 mM ethylenediamine tetraacetic acid (EDTA), pH 7.4. After 20 min. on ice, the lysate was centrifuged at 13,000 x g for 15 min at 4°C and treated with RNAse A (100 µg/ml at 37°C for 1 h). The supernatant containing degraded RNA and fragmented DNA, but not intact chromatin, was extracted with phenol chloroform. Nucleic acids were precipitated with 1 vol. of ethanol and 300 mM sodium acetate. Samples were electrophoresed in a 1.5% agarose gel and visualized by ethidium bromide staining.

Assessment of nuclear morphology. Cells were labeled with Hoechst 33258 (5 µg/ml) for 15 min, washed in PBS, and fixed in 4% formaldehyde. Fixed cells were washed and viewed on an Olympus IMT-2 inverted research microscope using the filter for 340 nm.

Data presentation and analysis. For statistical analysis a one-way or a two-way analysis of variance (ANOVA) was used to identify overall treatment effects, followed by the unpaired two-tailed Student's t-test for selective comparison of individual data groups. Bonferroni's correction was applied to the significance level. Only significances relevant for the discussion of the data are indicated in each figure.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of cultured cerebellar neurons to DTX-2 for 24 h resulted in neurotoxicity characterized first by the disintegration of the neurites and later by swelling of cell bodies and cellular death. Degeneration of the neuronal network started to become evident at DTX-2 concentrations of 2.5 nM (Fig. 1b), while after exposure to 5 nM DTX-2 all neurites were very weak or completely fragmented and cell bodies appeared rounded and swollen (Fig. 1c). The presence of vacuoles and cellular fragmentation was also evident. Neurotoxicity by DTX-2 was a long-term process since neither cell death nor morphological signs of toxicity were observed in neurons exposed to the toxin for 18 h (data not shown). It should be noted that despite the strong signs of toxicity observed in 5 nM DTX-2-treated cultures, a considerable number of cells (~50%) were still able to retain the vital dye fluorescein diacetate and therefore they were considered alive under this criterion (see Materials and Methods). Further increase in the toxin concentration did not produce significant changes in the neurotoxicity pattern and further decreased the number of neurons retaining fluorescein (Fig. 1d). The concentration of DTX-2 that produced a 50% reduction in maximum neuronal survival (EC50) was estimated at ~8 nM. In parallel experiments, the EC50 for okadaic acid neurotoxicity was ~2 nM, indicating an approximately 4-fold higher sensitivity of neurons to this toxin compared to DTX-2 (Fig. 1e).



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FIG. 1. Neurotoxic effect of DTX-2 in cerebellar neurons in culture. (a–d) Fluorescence photomicrographs of neuronal cerebellar cultures before (a) and after 24 h exposure to 2.5 nM (b), 5 nM (c), or 25 nM (d) dinophisistoxin-2 (DTX-2). Staining with the marker for living cells fluorescein diacetate showed cell bodies of live neurons and the neurites (medium arrows). At least some of the few number of astrocytes present in these cultures were also visible and appeared largely unaffected even at 25 nM DTX-2 (panel d, large arrow). Bare nuclei of dead neurons were visualized by staining with ethidium bromide (small arrows). (e) Percentage of live neurons per dish (mean ± SD, n = 4–6) after treatment of cultures for 24 h with DTX-2 or okadaic acid (OKA) at the indicated concentrations.

 
To investigate whether the action of DTX-2 involved the activation of biochemical pathways leading to apoptosis, we examined the DNA from cultured neurons exposed to DTX-2. Agarose gel electrophoresis of soluble DNA extracted from neurons treated with DTX-2 for 18 h revealed large DNA fragmentation characteristic of apoptotic cells, resulting from cleavage of nuclear DNA in internucleosomal regions (Wyllie, 1980Go). Fragmentation of DNA was a very early event in the death process. It was already evident in DNA extracted from neurons which had been exposed to DTX-2 for only 12 h, while at least 24 h were necessary to observe the clear morphological signs of toxicity and the significant decrease in neuronal survival reported in Figure 1. Based on the intensity of ethidium bromide staining, DNA from 12 h-treated neurons showed a quantitatively lower fragmentation than DNA from 18 h-treated neurons, whereas no soluble fragmented DNA was obtained from control neurons (Fig. 2a).



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FIG. 2. DTX-2 induces neuronal gene expression-dependent apoptosis. (a) Agarose gel electrophoresis of soluble DNA revealed considerable DNA fragmentation in neurons exposed to 25 nM DTX-2 for 12 h (lane 2) or 18 h (lane 3) but not in control neurons (lane 1). Size of the DNA molecular size markers (in base pairs): 9416, 6557, 4361, 2322, 2027, 560. (b–d) Photomicrographs of neuronal cultures unexposed (b) or exposed to 25 nM DTX-2 for 17 h in the absence (c) or in the presence (d) of the transcriptional inhibitor actinomycin D (1 µg/ml), and stained with the DNA-binding fluorochrome Hoechst 33258. In control cultures (b) neuronal nuclei appeared large in size and weakly stained (small arrows) compared to DTX-2-treated cultures, in which a considerable number of nuclei were smaller and contained brightly stained, condensed chromatin (arrowheads). Addition of actinomycin D prior to DTX-2 prevented the appearance of apoptotic nuclei in neurons exposed to DTX-2 (d).

 
Occurrence of apoptosis was further confirmed by the appearance of changes in the nuclear morphology of affected neurons. Staining with the DNA-binding fluorochrome Hoechst 33258 revealed that nuclei in DTX-2-treated cultures showed condensation or fragmentation of chromatin (Fig. 2c) compared to control cultures (Fig. 2b). The number of apoptotic nuclei in DTX-2-treated and untreated neurons was quantified and found to be 51 ± 4% (n = 6) and 6 ± 1% (n = 4), respectively.

Apoptosis can generally be inhibited by the suppression of gene expression. We used the transcriptional inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide to examine whether neuronal apoptosis induced by DTX-2 required newly synthesized proteins. No significant increase in the number of apoptotic nuclei was observed in neurons exposed to DTX-2 in the presence of actinomycin D (8 ± 2%, n = 6) compared to untreated neurons (6 ± 1%, n = 4) (Figs. 2b and 2d). Accordingly, both actinomycin D (1 µg/ml) and cycloheximide (5 µg/ml) prevented neurotoxicity by DTX-2 (Fig. 3).



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FIG. 3. Protein synthesis inhibitors inhibit neurotoxicity by DTX-2. Number of surviving neurons (mean ± SD, n = 4) before or after exposure of cerebellar neurons to 10 nM DTX-2 for 24 h, in the absence or in the presence of the transcriptional inhibitor actinomycin D (AcD, 1 µg/ml), or the protein synthesis inhibitor cyclohexymide (CHX, 5 µg/ml). CHX and AcD were added 1 h before DTX-2. *p < 0.01.

 
We noticed that morphology and viability of at least some of the few non-neuronal cells (~3%) present in the neuronal culture appeared mostly unaffected (see Fig. 1d) after exposure to DTX-2. In order to evaluate more precisely the selectivity of the neurotoxic effect of DTX-2 we prepared mixed neuroglial cultures containing neurons and astrocytes (see Materials and Methods). In these cultures non-neuronal cells, confirmed to be astrocytes by immunostaining with glial fibrillary acidic protein GFAP (data not shown), accounted for about 10% of the overall number of cells in culture and could be easily distinguished from neurons and counted. Also, mixed neuroglial cultures allow for reciprocal interaction between neurons and astrocytes, rendering it an appropriate experimental system to investigate a possible modulation by astrocytes of neurotoxicity by DTX-2. The results found in these experiments are represented in Figure 4. Compared to neurons, the effect of DTX-2 on glial cells required significantly higher concentrations of the toxin although, interestingly, damage appeared much earlier in these cultures. Thus, 7 h exposure to 50 nM DTX-2 (Fig. 4c) resulted in marked morphological changes of glial cells present in mixed cultures, which compared to untreated cultures (Fig. 4a) appeared less extended and spread, leaving large areas of the dish uncovered. In contrast, no significant alterations in morphology and survival were observed in co-cultured neurons after short (7 h) exposures to this high concentration (50 nM) of the toxin (Fig. 4c). Long exposures (24 h) to low (5 nM) concentrations of DTX-2 affected morphology and viability of neurons but not glial cells present in mixed cultures (Fig. 4d), while extensive toxicity of both neurons and glial cells were observed after 24 h exposure to 50 nM DTX-2 (Fig. 4e).



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FIG. 4. Differential toxicity of DTX-2 in cultured cerebellar neurons and astrocytes. (a–e) Fluorescence photomicrographs of cells grown in culture for 12 days taken before (a) or after exposure to 10 nM DTX-2 (b, d) or 50 nM DTX-2 (c, e) for 7 h (b, c) or 24 h (d, e). Neuronal somas and neurites appeared morphologically unaffected after short (7 h) exposures to DTX-2 (b, c, small arrows) independently of the concentration of toxin used. Long exposures (24 h) to low concentrations (10 nM) of DTX-2 caused degeneration of neurites and swelling of neuronal somas (panel d, large arrows) while morphology of astrocytes appeared mostly unaffected compared to untreated controls (see panel a). Glial cells exposed to 50 nM DTX-2 for 7 or 24 h showed condensation of cell bodies and retraction of processes (panel c and e, arrowheads). (f,g) Dose-response experiments for toxicity of neurons and astrocytes in mixed neuroglial cultures after exposure to DTX-2. Percentages of live glial cells (f) or live neurons (g) after treatment of mixed cultures with DTX-2 for 7 or 24 h at the indicated concentrations are represented.

 
Quantification of cell survival revealed no significant differences between the number of glial cells retaining fluorescein diacetate after 7 or 24 h exposure to different concentrations of DTX-2 (Fig. 4f). Significant toxicity started at 15 nM DTX-2 and 50% reduction of surviving glial cells was achieved after exposure to approximately 50 nM DTX-2 for 7 h (EC507) or to approximately 30 nM DTX-2 for 24 h (EC5024). It has to be considered that although survival quantification considered all glial cells retaining fluorescein diacetate to be alive independently of their morphology, most glial cells appeared shrunken and degenerated after 7 h exposure to 50 nM DTX-2 (see Fig. 4c). Dose-response experiments for co-cultured neurons in these mixed cultures, as the one represented in Fig. 4g, showed no significant changes in neuronal survival after 7 h exposure up to 50 nM DTX-2. As for 24 h exposure, concentration dependence, as well as maximal toxicity and EC50 values, were similar to those observed in pure neuronal cultures (see Figs. 4g and 1e).

The effect of DTX-2 on astrocyte morphology and viability was further confirmed by using highly enriched astroglial cultures containing no neurons. More than 80% of the nuclei visualized by ethidium bromide staining in these cultures corresponded to GFAP-positive cells (not shown). After 10 h exposure to 50 nM DTX-2, the majority of these astrocytes had already shrunk and degenerated (Fig. 5b), and DNA staining by Hoechst 33258 revealed that glial toxicity by DTX-2 occurred via apoptosis. Thus, 30 ± 7% (n = 8) of nuclei appeared either shrunken or degraded and with aggregation of chromatin (Fig. 5d) compared to 3 ± 2% (n = 4) in control cultures in which most nuclei had regular contours and were round and large in size (Fig. 5c).



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FIG. 5. DTX-2 induces apoptotic degeneration of astrocytes. Phase contrast (a, b) and Hoechst 33258 fluorescence (c, d) photomicrographs of highly enriched astroglial cultures before (a, c) or after (b, d) exposure to 50 nM DTX-2 for 10 h. Most nuclei of astrocytes exposed to DTX-2 appeared hypercondensed and irregularly shaped (arrows) or with extensive fragmentation of their chromatin (large arrows) compared to nuclei from untreated astrocytes which had regular contours and were round and large in size (arrowheads).

 
As apoptosis is often related to reactive oxygen species (ROS) production and oxidative injury, we assessed for the participation of oxidative stress in DTX-2-induced toxicity. We used the fluorescent probe carboxy-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA), as dichlorodihydrofluorescein and derivatives have proved extremely useful for assessing the overall oxidative stress in toxicological phenomena in a variety of experimental systems including cultured neurons (Behl, 1994Go; Fernández-Sánchez et al., 2001Go). No significant differences were found in fluorescence intensities in neurons exposed to 10 nM DTX-2 for 15–18 h or in astrocytes exposed to 50 nM for 6–8 h when compared to the corresponding untreated cultures (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The biological effects of the DSP-type dinophysistoxins have been poorly explored. Our results show that DTX-2 is a potent neurotoxin for cerebellar neurons in primary culture. Concentrations of DTX-2 in the low nanomolar range caused visible signs of neurotoxicity, including degeneration of neurites and swelling of cell bodies, leading to widespread neuronal death (Fig. 1). A similar neurotoxicity pattern has been previously reported for okadaic acid at concentrations as low as 0.5 nM in this experimental system (Fernández et al., 1991Go, 1993Go). The regulatory limit for DSP toxins in mussels has been established at 160 µg okadaic acid-equivalents/kg, equivalent to approximately 200 nM of DSP toxin. Thus, the effects of DTX-2 on neurons and astrocytes reported herein occurred at toxin levels far below the regulatory limits and suggest that prolonged exposures to subtoxic concentrations of this toxin may damage particularly vulnerable structures such as the nervous system. DTX-2 shares with okadaic acid the capacity to bind and inhibit type 1 and type 2A ser/thr phosphatases (Bialojan and Takai, 1988Go), leading to the stabilization of phosphorylated forms of proteins substrates of protein phosphatase 1 (PP1) and 2A (PP2A). The differential sensitivity to DTX-2 we observed between neuronal and non-neuronal cells is consistent with the idea that some of these proteins may be either specific for neurons or particularly important for neuronal functioning. Indeed, evidence exists supporting a major role for PP1 and PP2A in Alzheimer's disease and neurodegenerative pathologies associated with the abnormal hyperphosphorylation of microtubule-associated protein tau. Decreased PP1 and PP2A activities have been observed in Alzheimer's disease brains compared to age-matched controls (Gong et al., 1993Go). Moreover, a direct dephosphorylation of tau by PP2A has been demonstrated using hyperphosphorylated tau from Alzheimer's disease brain as substrate (Gong et al., 1995Go), as well as in rat forebrain neurons (Bennecib et al., 2000Go). It has to be considered that DTX-2 and okadaic acid often occur together in DSP blooms, and that residual levels of these toxins may persist in contaminated mussels for a long time even below the regulatory detection limits (Fernández et al., 1998Go). Also, the lipophilic nature of DTX-2 and okadaic acid may facilitate their accumulation in tissues, including the nervous tissue. Altogether, these observations suggest that chronic exposure to low amounts of these seafood toxins may involve a risk for human health that should be taken into consideration. This risk could be particularly relevant in certain periods of human life such as embryonic development or senescence.

Neurotoxicity by DTX-2 was accompanied by the laddering-like fragmentation of DNA, a hallmark of apoptosis (Raff, 1992Go), similarly to what has been previously observed for okadaic acid (Fernández-Sánchez et al., 1996Go). Assessment of chromatin condensation by staining of nuclei with the Hoechst dye further confirmed that neuronal degeneration by DTX-2 was attributable to apoptosis. DNA fragmentation occurred early in the death process, as it could be detected long before neuronal degeneration and death could be observed. Also, the observation that it could be prevented by inhibitors of RNA and protein synthesis indicates that apoptosis by DTX-2 is a genetically controlled process. These observations are consistent with a regulation by DTX-2 of expression of genes coding for proteins playing active roles in the control of apoptosis. Among possibilities, p53 and members of the bcl-2 family, Bcl-2, BAX, and bcl-x, appear to be plausible candidates as they have been suggested to be involved in the apoptotic response induced by okadaic acid in other cell types (Benito et al., 1997Go; Sheikh et al., 1996Go; Yan et al., 1997Go). Experiments are in progress to investigate the possible role for the expression of these genes in the neuronal apoptosis induced by DTX-2.

The rescue of neurons by the transcription inhibitor actinomycin D also raises the question of whether aberrant activation of cell cycle regulatory proteins could play a role in neurotoxicity by DTX-2, as demonstrated for apoptosis by other stimuli in post-mitotic neurons including cerebellar granule neurons (Martin-Romero et al., 2000Go; Padmanabhan et al., 1999Go; Park et al., 1996Go). We are currently investigating the possible involvement of cyclin-dependent kinases in the effects of DTX-2. Interestingly, preliminary data showed that in glial cells apoptosis by DTX-2 could not be inhibited by actinomycin D (data not shown). Thus, comparison between neuronal and glial cells may provide new insights about the mechanisms controlling apoptosis in neuronal versus non-neuronal cells and these cerebellar cultures appear to be an appropriate model for these types of studies.

Recent data suggest that toxic insults to glial cells might cause degeneration of surrounding neurons both in experimental systems and in human pathologies (Akiyama et al., 2000Go; Ekdahl et al., 2003Go). Consistently, we have previously reported that apoptotic degeneration of cultured cerebellar glial cells due to long exposures to aluminum induced neuronal degeneration and death (Suárez-Fernández et al., 1999Go). In contrast, DTX-2 toxicity appeared to occur independently in neurons and glial cells. The relative resistance of neurons to short exposures of high concentrations of DTX-2 compared to the co-cultured astrocytes, suggests that DTX-2-induced glial toxicity is not likely to be associated either with increased glial reactivity toward neuronal cells or with the release of neurotoxic factors from glial cells. Preliminary experiments indicate a similar pattern of neuro-glial toxicity for okadaic acid (data not shown). Thus, these DSP toxins may represent very interesting biochemical tools for elucidating how glial cells may die without affecting neighboring neurons.

We have previously proposed a neuronal bioassay (García-Rodríguez et al., 1998Go) for the detection of DSP toxins, based in the high sensitivity of cultured cerebellar neurons to okadaic acid (Fernández et al., 1991Go, 1993Go). By using purified okadaic acid as reference standard, neuronal bioassay proved to be a suitable method for the detection and quantification of DSP toxicity in toxic mussel extracts which were demonstrated by HPLC to contain both okadaic acid and DTX-2, and allowing for a detection limit as low as 20 µg okadaic acid equivalents per kg of fresh animal tissue. Results described herein further support the suitability of cultured neurons for the analysis of total DSP toxicity. It is worth noting that DTX-2 appeared to be a less potent neurotoxin than okadaic acid (Fig. 1). Thus, cultured cerebellar neurons provide a sensitive model for the evaluation of DSP toxins that may give also useful information about the relative potency of toxins. In view of the marked effects of DTX-2 on astrocytes we report, and given that these cells can be of much more easily handling and commercialization than cultured neurons, cultured astrocytes also represent a very promising model for the biological detection of DSP toxins.


    ACKNOWLEDGMENTS
 
We thank Dr. A. Sampedro and Dr. A. M. Nistal from the Image Process Service of the University of Oviedo for the use of the laser confocal microscope. We also thank Dr. M. D. Paz-Caballero from the Psychology Department of the University of Oviedo for her help with the statistical analysis. This work was supported by CICYT, Grants SAF94-0394 and MCT-01-REN2959-C0404.


    NOTES
 

1 To whom correspondence should be addressed at Antonello Novelli or María-Teresa Fernández-Sánchez, Departamento de Bioquímica y Biología Molecular, Edificio "Santiago Gascón". Campus "El Cristo", Universidad de Oviedo, 33006 Oviedo, Spain. Fax: (+34) 985103157. E-mail: neurolab{at}bioquimica.uniovi.es.


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 DISCUSSION
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