Diarrheic Shellfish Poisoning Research Articles

Experimental Basis for the High Oral Toxicity of Dinophysistoxin 1: A Comparative Study of DSP

Okadaic acid (OA) and its analogues, dinophysistoxin 1 (DTX1) and dinophysistoxin 2 (DTX2), are lipophilic and heat-stable marine toxins produced by dinoflagellates, which can accumulate in filter-feeding bivalves. These toxins cause diarrheic shellfish poisoning (DSP) in humans shortly after the ingestion of contaminated seafood. Studies carried out in mice indicated that DSP poisonous are toxic towards experimental animals with a lethal oral dose 2–10 times higher than the intraperitoneal (i.p.) lethal dose. The focus of this work was to study the absorption of OA, DTX1 and DTX2 through the human gut barrier using differentiated Caco-2 cells. Furthermore, we compared cytotoxicity parameters. Our data revealed that cellular viability was not compromised by toxin concentrations up to 1 μM for 72 h. Okadaic acid and DTX2 induced no significant damage; nevertheless, DTX1 was able to disrupt the integrity of Caco-2 monolayers at concentrations above 50 nM. In addition, confocal microscopy imaging confirmed that the tight-junction protein, occludin, was affected by DTX1. Permeability assays revealed that only DTX1 was able to significantly cross the intestinal epithelium at concentrations above 100 nM. These data suggest a higher oral toxicity of DTX1 compared to OA and DTX2.

Impact of economic development on the dynamics of phytoplankton and physic-chemical quality of Dakhla Bay (South of Morocco)

A detailed monitoring of physic-chemical parameters and a complete inven-torization of phytoplankton species was conducted from May 2011 to May 2012 in Dakhla bay in south of Morocco. This study comes to bridge the gap of environmental data which has started since 2000 by Moroccan national institute of fisheries research (INRH) in this area. The results showed a differ-ence in the distribution of physic-chemical parameters (especially nutrient and dissolved oxygen). In addition, inventory of phytoplankton species has allowed identifying three different groups: diatoms (Rhizosolenia spp, Lepto-cylindrus spp, Thalassionema spp, Pseudo- nizschia spp.,...) represented 92% of the total phytoplankton flora, dinoflagellates (Scrippsiella sp, Prorocen-trum sp, Gymnodinium sp.,...) with 7%, while Silicoflagellates (Dictyocha sp) contributed with 1%. Seasonal fluctuation of these groups was governed, in first order, by the importance of upwelling in the region during the summer (intense phytoplankton activity). Yet this activity was much stronger in the PK25 site for tourism activity that suggested a remarkable anthropogenic input. An occurrence of Scrippsiella species (45% Hoja Iliamera growing area) and Prorocentrum sp (41% Duna Blanca aquaculture area), recognized as species of colored waters, was permanent at the study sites. Alexandrium sp represented 1% of the total dinoflagellates in Duna Blanca (<1% in the other sites), whereas Dinophysis sp (<1%) was observed in Lassarga (the entrance bay) and PK25 only. The latter two species are potentially the cause of PSP toxins's production (Paralytic Shellfish Poisoning) and DSP (Diarrheic Shellfish Poisoning) source of contamination of bivalves in the Bay during 2004 and 2006.

Okadaic Acid: More than a Diarrheic Toxin

Okadaic acid (OA) is one of the most frequent and worldwide distributed marine toxins. It is easily accumulated by shellfish, mainly bivalve mollusks and fish, and, subsequently, can be consumed by humans causing alimentary intoxications. OA is the main representative diarrheic shellfish poisoning (DSP) toxin and its ingestion induces gastrointestinal symptoms, although it is not considered lethal. At the molecular level, OA is a specific inhibitor of several types of serine/threonine protein phosphatases and a tumor promoter in animal carcinogenesis experiments. In the last few decades, the potential toxic effects of OA, beyond its role as a DSP toxin, have been investigated in a number of studies. Alterations in DNA and cellular components, as well as effects on immune and nervous system, and even on embryonic development, have been increasingly reported. In this manuscript, results from all these studies are compiled and reviewed to clarify the role of this toxin not only as a DSP inductor but also as cause of alterations at the cellular and molecular levels, and to highlight the relevance of biomonitoring its effects on human health. Despite further investigations are required to elucidate OA mechanisms of action, toxicokinetics, and harmful effects, there are enough evidences illustrating its toxicity, not related to DSP induction, and, consequently, supporting a revision of the current regulation on OA levels in food.

SPR immunosensor for the detection of okadaic acid in mussels using magnetic particles as antibody carriers

Protein G-coated magnetic particles (MPs) were used as immobilisation supports for an antibody against okadaic acid (MAbOA) and carriers into a surface plasmon resonance (SPR) device for the development of a direct competitive immunosensor for okadaic acid (OA). SPR analysis of MAbOA–MP conjugates demonstrated that conjugations were successful with complete immobilisation of all the antibody biomolecules onto the MPs. Moreover, MAbOA–MP conjugates provided up to 11-fold higher SPR signals, compared to free MAbOA. The use of conjugates in the direct competition assay provided a 3-fold lower LODμg/L (2.6μg of OA/L, equivalent to 12μg of OA/kg mussel meat). The presence of mussel matrix did not interfere in the OA quantification as seen in the calibration curves. Mussel samples, obtained from Ebro Delta's bays (NW Mediterranean) during a diarrheic shellfish poisoning (DSP) event and in the presence of Dinophysis sacculus, an OA producer, in the shellfish production area, were analysed with the MP-based SPR immunosensor. The OA contents correlated with those obtained by liquid chromatography–tandem mass spectrometry (LC–MS/MS) (y=0.984x−5.273, R2=0.789, p<0.001) and by mouse bioassay (MBA).

Multidetection of Paralytic, Diarrheic, and Amnesic Shellfish Toxins by an Inhibition Immunoassay Using a Microsphere-Flow Cytometry System

The presence of paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP) toxins in seafood is a severe and growing threat to human health. In order to minimize the risks of human exposure, the maximum content of these toxins in seafood has been limited by legal regulations worldwide. The regulated limits are established in equivalents of the main representatives of the groups: saxitoxin (STX), okadaic acid (OA), and domoic acid (DA), for PSP, DSP, and ASP, respectively. In this study a multidetection method to screen shellfish samples for the presence of these toxins simultaneously was developed. Multiplexing was achieved using a solid-phase microsphere assay coupled to flow-fluorimetry detection, based on the Luminex xMap technology. The multidetection method consists of three simultaneous competition immunoassays. Free toxins in solution compete with STX, OA, or DA immobilized on the surface of three different classes of microspheres for binding to specific monoclonal antibodies. The IC50 obtained in the buffer was similar in single- and multidetection: 5.6 ± 1.1 ng/mL for STX, 1.1 ± 0.03 ng/mL for OA, and 1.9 ± 0.1 ng/mL for DA. The sample preparation protocol was optimized for the simultaneous extraction of STX, OA, and DA with a mixture of methanol and acetate buffer. The three immunoassays performed well with mussel and scallop matrixes displaying adequate dynamic ranges and recovery rates (around 90% for STX, 80% for OA, and 100% for DA). This microsphere-based multidetection immunoassay provides an easy and rapid screening method capable of detecting simultaneously in the same sample three regulated groups of marine toxins.

Effect of okadaic acid on carpet shell clam (Ruditapes decussatus) haemocytes by in vitro exposure and harmful algal bloom simulation assays

Okadaic acid (OA), produced by dinoflagellates during harmful algal blooms (HAB), belongs to the Diarrheic Shellfish Poisoning toxins that cause gastrointestinal symptoms in humans after consumption. In the present work, Ruditapes decussatus haemocytes were selected to evaluate the effect of OA on cell viability, enzymatic status and immune capacity through the measure by flow cytometry of apoptosis-cell death, non-specific esterase activity and phagocytosis. In order to compare different exposure conditions, two experiments were developed: in vitro exposure to OA and HAB simulation by feeding clams with the OA producer, Prorocentrum lima. Apoptosis was not OA dose-dependent and cell death increased in both assays. Phagocytosis of latex beads and esterase activity decreased in haemocytes incubated with OA. In contrast, esterases increased during the feeding with P. lima. Our results showed that OA and the simulated HAB caused damages on haemocyte functions and viability.

The marine toxin okadaic acid induces alterations in the expression level of cancer-related genes in human neuronal cells

Okadaic acid (OA) is one of the most common and highly distributed marine toxins. It can be accumulated in several molluscs and other marine organisms and cause acute gastrointestinal symptoms after oral consumption by humans, called diarrheic shellfish poisoning. However other toxic effects beyond these gastrointestinal symptoms were also reported. Thus, OA was found to induce important chromosomal abnormalities and other genetic injuries that can lead to severe pathologies, including cancer. Furthermore, the relationship between OA and carcinogenic processes has been previously demonstrated in in vivo studies with rodents, and also suggested in human epidemiological studies. In this context, further research is required to better understand the underlying mechanisms of OA-related tumourigenesis. In a previous study, we identified 247 genes differentially expressed in SHSY5Y neuroblastoma cells exposed to 100nM OA at different times (3, 24 and 48h) by means of suppression subtractive hybridization. These genes were involved in relevant cell functions such as signal transduction, cell cycle, metabolism, and transcription and translation processes. However, due to the high potential percentage of false positives that may be obtained by this approach, results from SSH are recommended to be analyzed by an independent method. In the present study, we selected ten genes related to cancer initiation or progression, directly or indirectly, for further quantitative PCR analysis (ANAPC13, PTTG1, CALM2, CLU, HN1, MALAT1, MAPRE2, MLLT11, SGA-81M and TAX1BP1). Results obtained showed important alterations in the expression patterns of all the genes evaluated at one or more treatment times, providing, for the first time, a possible explanation at the molecular level of the potential relationship between the consumption of OA-contaminated shellfish and the incidence of different cancers in humans. Nevertheless, given the complexity of this process, more exhaustive studies are required before drawing any final conclusion.

Inhibition Equivalency Factors for Dinophysistoxin-1 and Dinophysistoxin-2 in Protein Phosphatase Assays: Applicability to the Analysis of Shellfish Samples and Comparison with LC-MS/MS

The protein phosphatase inhibition assay (PPIA) is a well-known strategy for the determination of diarrheic shellfish poisoning (DSP) lipophilic toxins, which deserves better characterization and understanding to be used as a routine screening tool in monitoring programs. In this work, the applicability of two PPIAs to the determination of okadaic acid (OA), dinophysistoxin-1 (DTX-1), dinophysistoxin-2 (DTX-2), and their acyl ester derivatives in shellfish has been investigated. The inhibitory potencies of the DSP toxins on a recombinant and a wild PP2A have been determined, allowing the establishment of inhibition equivalency factors (IEFs) (1.1 and 0.9 for DTX-1, and 0.4 and 0.6 for DTX-2, for recombinant and wild PP2A, respectively). The PPIAs have been applied to the determination of OA equivalent contents in spiked and naturally contaminated shellfish samples. Results have been compared to those obtained by LC-MS/MS analysis, after application of the IEFs, showing good agreements.

Determination of okadaic acid related toxins from shellfish (&amp;lt;i&amp;gt;sinonovacula constricta&amp;lt;/i&amp;gt;) by high performance liquid chromatography tandem mass spectrometry

Consumption of shellfish contaminated with algal toxins produced by marine dinoflagellates can lead to diarrhetic shellfish poisoning (DSP). It was therefore essential that there are analytical techniques to identify and quantify DSP toxins in shellfish. This new methodology could facilitate DSP monitoring and create a means of rapidly responding to incidents threatening public health. In the last years there were different analytical methods for DSP, such as mouse bioassay and LC-FLD. With the development of instrument, Liquid chromatography-mass spectrometry was substituted for other analytical methods with its good sensitivity and selectivity and without derivatization for the determination of DSP. In this report, a high performance liquid chromatogra-phytandem mass spectrometric（HPLC-MS/MS）method was developed for the simultaneous determination of okadaic acid (OA) and dinophysistoxins(DTX1) in Sinonovacula constricta. Optimization of pretreatment experiment was carried out to maximize recoveries and the effectiveness. The analytes were determined under multi-reactions monitoring (MRM) scan type with tandem mass analyzer using negative ion electrospray ionization (-ESI) mode .Finally, the detection and identification of OA and DTX-1 were based upon their retention times (RT) and the fragmentation patterns of their mass spectra. The method of LOQ for the two poisons was 0.02 mg·kg-1.The real sample test showed that this method could be used for sensitive, fast, and accurate determination of the two diarrheic shellfish poisons in shellfish.

Microbiological and Marine Biotoxins Contamination of Raw Bivalve Molluscs Commercially Available in Poland

Abstract The study was carried out on live bivalve molluscs available on Polish market. Microbiological tests were performed for the presence of Salmonella sp., Vibrio parahaemolyticus, spore-forming anaerobe bacteria, and coagulase-positive Staphylococcus sp., and for the enumeration of Escherichia coli. ELISA was used for the determination of marine biotoxins, paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP), and diarrhoeic shellfish poisoning (DSP). Microbiological examinations were performed according to ISO and Polish Standards. Salmonella sp. was not detected in any sample tested. Coagulase-positive staphylococci were identified in 9.0% of the samples. V. parahaemolyticus was isolated from 17.0% of mussels. Shellfish were highly contaminated by anaerobes, which were isolated from 68.0% of the samples. The number of E. coli ranged from &lt;2.0 x 101 up to &gt;1.8 x 104 MPN/100 g. The majority of mussels were free from the marine biotoxins tested or contained them bellow the permitted level. The analysis of microbiological and toxicological status of raw bivalve molluscs available on Polish market indicates that they are generally safe for the consumers.

Gene expression profiling of human liver carcinoma (HepG2) cells exposed to the marine toxin okadaic acid

The marine toxin, okadaic acid (OA) is produced by dinoflagellates of the genera Prorocentrum and Dinophysis and is the causative agent of the syndrome known as diarrheic shellfish poisoning. In addition, OA acts as both a tumor promoter, attributed to OA-induced inhibition of protein phosphatases as well as an inducer of apoptosis. To better understand the potentially divergent toxicological profile of OA, the concentration-dependent cytotoxicity and alterations in gene expression on the human liver tumor cell line HepG2 upon OA exposure were determined using RNA microarrays, DNA fragmentation, and cell proliferation assays as well as determinations of cell detachment and cell death in different concentrations of OA. mRNA expression was quantified for approximately 15,000 genes. Cell attachment and proliferation were both negatively correlated with OA concentration. Detached cells displayed necrotic DNA signatures but apoptosis also was broadly observed. Data suggest that OA has a concentration dependent effect on cell cycle, which might explain the divergent effects that at low concentration OA stimulates genes involved in the cell cycle and at high concentrations it stimulates apoptosis.

Effects of okadaic acid on haemocytes from Mytilus galloprovincialis: A comparison between field and laboratory studies

Individuals of Mytilus galloprovincialis, contaminated with Diarrheic Shellfish Poisoning (DSP) toxins, were studied with the aim to correlate the okadaic acid (OA) body burden and the percentage of damaged haemocytes by quantifying annexin V positive cells by flow cytometry. Results showed less percentage of damaged haemocytes in high OA contaminated samples. These data were compared with results of in vitro assays of mussel haemocytes exposed to increased concentrations of OA. Similarly, haemocytes exposed to the most concentrated OA solution were less damaged.

Production of monoclonal antibody and application in indirect competitive ELISA for detecting okadaic acid and dinophytoxin-1 in seafood

Okadaic acid (OA) and analogues of dinophysistoxin (DTX) are key diarrheic shellfish poisoning (DSP) toxins, which possibly arouse DSP symptoms by consuming the contaminated shellfish. Because of the stable toxicity in high temperature and the long-term carcinogenicity, the outbreaks of DSP related to consumption of bivalve mollusks contaminated by DSP toxins pose a hazard to public health. Therefore, it is worth developing a fast and reliable analytical method for the detection of OA and analogues in shellfish. In this paper, an indirect competitive enzyme-linked immunosorbent assay (ELISA) (icELISA) for detecting OA and DTX-1 in seafood was developed based on monoclonal antibody (McAb). The OA was conjugated to human immunoglobulin G (IgG) and bovine serum albumin (BSA) by the active ester method as the immune antigen and the detective antigen. The spleen cells from BALB/c mice immunized with OA-IgG were fused with SP2/0 myeloma cells. A hybridoma cell line, which secreted McAb against OA, was selected by "limiting dilution" cloning. An icELISA was developed based on immobilized conjugate (OA-BSA) competing the McAb with the free OA in seafood sample. A hybridoma cell line, which secreted IgG1 subclass monoclonal antibody (McAb) against OA, was selected. The IC(50) of the McAb for OA and dinophytoxin-1 (DTX-1) were 4.40 and 3.89ng/mL, respectively. Based on the McAb, an indirect competitive ELISA for detection of OA and DTX-1 in seafood was developed. The regression equation was y = 54.713x - 25.879 with a coefficient correlation of R (2) = 0.9729. The linear range and the limit of detection were 0.4-12.5 and 0.45ng/mL, respectively. The average recovery of OA and DTX-1 spiked shellfish was 82.29% with the coefficient of variation of 7.67%. The developed icELISA is a fast, sensitive, and convenient assay for detecting of total amount of OA and DTX-1 in seafood.

Development of a Capillary Electrophoresis-Based Enzyme Immunoassay with Electrochemical Detection for the Determination of Okadaic Acid and Dinophysistoxin2 in Shellfish Samples

A capillary electrophoresis-based enzyme immunoassay (CE-EIA) with electrochemical (EC) detection system was developed for the determination of two diarrheic shellfish poisoning (DSP) toxins okadaic acid (OA) and dinophysistoxin2 (DTX2). In this method, after the competitive immunoreaction in liquid phase, the horseradish peroxidase (HRP)-labeled antigen (Ag*) and the bound enzyme-labeled complex (Ag*-Ab) were separated and then the system of HRP catalyzing H2O2/o-aminophenol (OAP) reaction was adopted. The limit of detection (S/N = 3) was determined to be 0.05 and 0.07 ng/mL for OA and DTX2, respectively. The total analysis time was less than 40 min. The developed CE-EIA with EC detection system was capable of quantitatively detecting OA and DTX2 contents in the tested contaminated samples, and the results were compared with the same samples analyzed through enzyme-linked immunosorbent assay (ELISA). Consistent results between CE-EIA with EC detection and ELISA were found in most of the tested samples. The proposed system appeared to be more sensitive and faster than ELISA for determination of OA and DTX2 in shellfish meat extracts. Real shellfish samples were validated in recovery test, and the recoveries tested by the proposed method were 91.7–108.3% and 95.2–112.5% for OA and DTX2, respectively. The CE-EIA with EC detection provides a valid and sensitive analytical approach, not previously available, for the determination of OA and DTX2 in shellfish samples.

Synthesis of artificial antigen and preparation of polyclonal antibody against okadaic acid

Okadaic acid(OA)is one kind of lipophilic marine biotoxins,and main pathogenic factor of diarrheic shellfish poisoning(DSP).In this paper,the artificial antigen was made by coupling hapten of OA with keyhole limpet hemocyanin(KLH)using active ester method,and the artificial antigen was tested by SDS-polyacrylamide gel electrophoresis and IR spectrum.After being obtained from rabbits,the antiserum was purified by Chromatography on Protein A Sepharose CL-4B gel column,and the purification efficiency was verified by ultraviolet scanning and ciELISA.The results showed the artificial antigen was prepared successfully and lots of interfering substances were removed.After being purified,the concentration of antiserum was 2.16 mg/mL,it had high titer(12 800)and sensitivity(the value of IC15 was 3.41 ng/mL),which was tested by ciELISA.The success of obtaining polyclonal antibody against OA with high titer and affinity provides the solid foundation which is practical and theoretical for developing rapid and economical detections.

Blooms of Azadinium cf. spinosum Elbrächter et Tillmann (Dinophyceae) in northern shelf waters of Argentina, Southwestern Atlantic

Azadinium spinosum has been identified as a species producer of azaspiracids (AZAs), marine toxins reported to cause human poisoning etiologically similar to diarrheic shellfish poisoning. AZAs are accumulated by molluscs via food web which, in turn, may cause human poisoning after consumption of contaminated shellfish. In this study, the presence of a species of the genus Azadinium, A. cf. spinosum, is reported for the first time in the Southwest Atlantic ocean, and two blooms in northern shelf waters of Argentina are analysed. Both blooms occurred during spring in succesive years and developed along extensive areas (up to 60km transection) of the middle shelf and near the shelf break in 1990, and also near the shelf break during 1991. The extended shelf break front of the Argentine Sea developed during spring and summer is a highly productive area rich in exploited marine resources. In November 1990, a first bloom (up to 9.03×106cellsL−1) produced heavy discolorations, and morphological and taxonomical studies showed that the causing organism was a new dinoflagellate genus. During September 1991 plankton composition and abundance along a transect from the coast to the Malvinas Current were analysed. Results showed that this dinoflagellate was the most important species in middle shelf waters, with high biomass and abundance values (up to 3×106cellsL−1) surpassing those of various chain-forming diatom Thalassiosira species, typical of the spring bloom. The description of this new taxon, according to the thecal analysis performed at that time by light and scanning electron microscopy, corresponds with little morphological differences to the thecal tabulation, cell morphology and dimensions of the recently described Azadinium spinosum. Taking into account these minor differences and due to the lack of both DNA sequence data and AZAs production, we decided to name it A. cf. spinosum. The phagotrophic dinoflagellate Gyrodinium fusus has shown to be an important grazer of A. cf. spinosum during the first bloom, suggesting that its grazing activity could have acted as a modeling factor in population levels of its prey. The presence of A. cf. spinosum in this area opens several questions as that related to toxin production given the regional importance of bivalve commercial fisheries.

Love wave biosensor for real-time detection of okadaic acid as DSP phycotoxin

This paper reports the detection of okadaic acid (OA) as a Diarrheic Shellfish Poisoning (DSP) toxin with an acoustic wave platform in real-time. According to the FDA, the threshold for safe consumption of shellfish is 20μg of OA for 100g of shellfish tissue. The high gravimetric sensitivity of Love wave acoustic devices allow, in liquid media, immuno-detection thanks to immobilized specific antibodies. The biosensor is composed of two lines, one for the test and one used as a reference. Sensitive films were deposited through a PolyDiMethylSiloxane (PDMS) microfluidic chip. On both lines, anti-okadaic acid antibodies (OA-Ab) and saturating agents were successively injected under continuous flow with controlled flow rate. On the test line, okadaic acid (OA) was injected while an unspecific peptide (6×Histidine, 6×His) was used on the control line. On both lines, polyclonal OA-Ab were re-injected a second time to reveal previously fixed OA on the test line. Measured frequency shifts were three times higher on test lines than on control lines. In these conditions, for only 2μg of OA used for detection, the acoustic wave platform could detect DSP toxins with only 10g of shellfish tissue.

Evaluation of Genotoxicity in Gills and Hemolymph of Clam Ruditapes decussatus Fed with the Toxic Dinoflagellate Prorocentrum lima

Diarrheic shellfish poisoning (DSP) is a gastrointestinal (GIT) disease that appears a few hours after ingesting okadaic acid (OA)-contaminated mollusks; okadaic acid is present in dinoflagellates of the genera Dinophysis and Prorocentrum. Toxic manifestations occur all year round at a higher or lesser intensity, and as a consequence, extractive production factories need to be closed during these periods which affects the economy of aquaculture industries. Although the concentration of harmful algae is usually found at high levels in clam digestive gland, bivalve mortality was not increased. In this study, the genotoxic effects produced by OA in clam Ruditapes decussatus were determined using the comet assay. In vitro (exposing hemocytes to different concentrations of OA) and in vivo (feeding clams with toxic dinoflagellate Prorocentrum lima) experiments were conducted in order to determine the genotoxic effects of OA on bivalve cells. Hemocytes and gill cells were analyzed by in vivo and in vitro approaches. While the in vitro study showed a rapid effect of OA on hemocytes, data obtained in the in vivo experiment reflected contradictory results dependent upon the concentration of OA and cell type evaluated. An increase in DNA damage was observed at the lower concentration and only in gill tissue. The results obtained may contribute to a better understanding of the mechanisms underlying genotoxic effects induced by OA on bivalves.

Evaluation of Okadaic Acid-Induced Genotoxicity in Human Cells Using the Micronucleus Test and γH2AX Analysis

Marine algal blooms have become a public health concern due to increasing frequency in the environment and severity of exposure consequences. Human intoxications produced by phycotoxins occur globally through consumption of marine fish products containing bioaccumulated toxins. Okadaic acid (OA) is the main representative of diarrheic shellfish poisoning (DSP) toxin. OA was found to inhibit protein phosphatases and to produce oxidative damage, as well as to disturb different cellular functions including cell cycle, gene expression, and DNA repair mechanisms. The aim of this study was to determine whether OA induced genotoxicity by using a micronucleus (MN) test and γH2AX analysis, and to elucidate the underlying mechanisms. Human peripheral blood leukocytes, neuroblastoma cells (SHSY5Y), and hepatoma cells (HepG2) were treated with a range of OA concentrations in the presence and absence of S9 fraction. MN induction was observed in leukocytes at all concentrations tested, and in SHSY5Y and HepG2 cells only at the highest concentration (1000 nM). In contrast, γH2AX analysis was only positive for HepG2 cells. Taking together these data, in addition to the comet assay results obtained in a previous study in this issue, OA was found to exert a either a clastogenic or aneugenic effect dependent upon the cell types examined.

Induction of oxidative DNA damage by the marine toxin okadaic acid depends on human cell type

The marine toxin okadaic acid (OA) is the main representative of diarrhoeic shellfish poisoning (DSP) toxins. Its ingestion induces nausea, vomiting, diarrhoea and abdominal ache. It has also been found to trigger cellular and molecular effects at low concentrations. Its mechanism of action has not been described yet. Results of a previous study showed that OA can induce cytotoxic and genotoxic effects, both directly and indirectly, and modulations in DNA repair processes in three different types of human cells (leukocytes, SHSY5Y neuroblastoma and HepG2 cells). These effects varied depending on the type of cell and the concentration employed ( Valdiglesias et al., 2010). On that basis, the ability of OA to induce oxidative DNA damage on the same cell types was investigated in the present study. To this end, the antioxidant enzymes catalase and N-acetylcysteine, and the human DNA- glycosylase hOGG1 were used in combination with the alkaline Comet assay. The cells were treated with a range of OA concentrations (5–1000 nM) in the presence and absence of S9 fraction. The results of this study showed that OA induces oxidative DNA damage directly in leukocytes, directly and indirectly in SHSY5Y cells, while it does not induce oxidative DNA damage in HepG2 cells. Combining the outcomes of both studies, the data showed that OA induces both cytotoxicity and genotoxicity, including DNA strand breaks and oxidative DNA damage, in the cells evaluated. However, the extent of these effects are cell type dependent.