Toxicity and differential oxidative stress effects on zebrafish larvae following exposure to toxins from the okadaic acid group.

Okadaic acid-group (OA-group) is a set of lipophilic toxins produced only in seawater by species of the Dinophysis and Prorocentrum genera, and characterized globally by being associated with harmful algal blooms (HABs). The diarrhetic shellfish poisoning toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1) are the most prevalent toxic analogues making up the OA-group, which jeopardize environmental safety and human health through consumption of hydrobiological organisms contaminated with these toxins that produce diarrhetic shellfish poisoning (DSP) syndrome in humans. Consequently, a regulatory limit of 160 µg of OA-group/kg was established for marine resources (bivalves). The aim of this study was to investigate effects varying concentrations of 1-15 µg/ml OA or DTX-1 on toxicity, development, and oxidative damage in zebrafish larvae (Danio rerio). After determining the lethal concentration 50 (LC50) in zebrafish larvae of 10 and 7 µg/ml (24 h) and effective concentration 50 (EC50) of 8 and 6 µg/ml (24 h), different concentrations (5, 6.5, or 8 µg/ml of OA and 4, 4.5, or 6 µg/ml of DTX-1) were used to examine the effects of these toxins on oxidative damage to larvae at different time points between 24 and 120 hpf. Macroscopic evaluation during the exposure period showed alterations in zebrafish including pericardial edema, cyclopia, shortening in the anteroposterior axis, and developmental delay. The activity levels of biochemical biomarkers superoxide dismutase (SOD) and catalase (CAT) demonstrated a concentration-dependent decrease while glutathione peroxidase (GPx) and glutathione reductase (GR) were markedly elevated. In addition, increased levels of oxidative damage (malondialdehyde and carbonyl content) were detected following toxin exposure. Data demonstrate that high concentrations of OA and DTX-1produced pathological damage in the early stages of development <48 h post-fertilization (hpf) associated with oxidative damage.

In vitro biotransformation of OA-group and PTX-group toxins in visceral and non-visceral tissues of Mytilus chilensis and Ameghinomya antiqua.

Lipophilic marine toxins (LMTs) are made up of multiple groups of toxic analogues, which are characterised by different levels of cellular and toxic action. The most prevalent groups in the southern Pacific zone are: a) okadaic acid group (OA-group) which consists of okadaic acid (OA) and dinophysistoxin-1 (DTX-1); and, b) pectenotoxin-2 (PTX2) group which consists of pectenotoxin-2 (PTX-2). The main objective of our study was to examine in vitro biotransformation of OA-group and PTX-group in the tissues of two endemic species of bivalves from southern Chile; blue mussels (Mytilus chilensis) and clams (Ameghinomya antiqua). The biotransformation processes of both groups were only detected in the digestive glands of both species using LC-MS/MS. The most frequently detected analogues were acyl derivatives (≈2.0 ± 0.1 µg ml-1) for OA-group and PTX-2SA (≈1.4 ± 0.1 µg ml-1) for PTX-group, with a higher percentage of biotransformation for OA-group (p < .001). In addition, simultaneous incubations of the different analogues (OA/PTX-2; DTX-1/PTX-2 and OA/DTX-1/PTX-2) did not show any interaction between the biotransformation processes. These results show that the toxicological variability of endemic species leads to biotransformation of the profile of toxins, so that these new analogues may affect people's health.

Toxins of Okadaic Acid-Group Increase Malignant Properties in Cells of Colon Cancer.

Diarrhetic shellfish poisoning (DSP) is a syndrome caused by the intake of shellfish contaminated with a group of lipophilic and thermostable toxins, which consists of okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2). These toxins are potent protein Ser/Thr phosphatase inhibitors, mainly type 1 protein phosphatase (PP1) and type 2A protein phosphatase (PP2A). Different effects have been reported at the cellular, molecular and genetic levels. In this study, changes in cell survival and cell mobility induced by OA, DTX-1 and DTX-2 were determined in epithelial cell lines of the colon and colon cancer. The cell viability results showed that tumoral cell lines were more resistant to toxins than the nontumoral cell line. The results of the functional assays for testing cell migration, evaluation of cell death and the expression of proteins associated with cell adhesion showed a dual effect of toxins since in the nontumoral cell line, a greater induction of cell death, presumably by anoikis, was detected. In the tumoral cell lines, there was an induction of a more aggressive phenotype characterized by increased resistance to toxins, increased migration and increased FAK activation. In tumoral cell lines of colon cancer, OA, DTX-1/DTX-2 induce a more aggressive phenotype.

High amount of dinophysistoxin-3 in Mytilus chilensis collected in Seno de Reloncaví, chile, during massive human intoxication associated with outbreak of Vibrio parahaemolyticus.

This study describes the detection of high amount of 7-O-acyl-derivative dinophysistoxin-1 (Dinophysistoxin-3) in filter bivalves collected on February 2005 in the Seno de Reloncaví, Puerto Montt City, Southern Chile, in the same period of time where an intoxication episode was associated with the presence of Vibrio parahaemolyticus in shellfish. The Diarrhetic Shellfish Poisoning (DSP) mouse bioassay of mussel extract samples, performed as described for regulatory testing, were negative to DSP toxins. Therefore, the same mussel samples collected from 8 places of Seno de Reloncaví were then analyzed by the HPLC-FLD method with pre-column derivatization procedure for DSP toxins. The samples showed mainly 7-O-acyl-derivative dinophysistoxin-1 (Dinophysistoxin-3) in concentrations ranging from 190.3 +/- 6.8 to 311.1 +/- 4.8 ng of DSP toxin/g hepatopancreas and less amounts of Dinophysistoxin-1 ranging from 1.9 +/- 1.5 to 11.7 +/- 4.6 ng of DSP toxin/g hepatopancreas. After alkaline hydrolysis of the mussel extracts, 279.4 +/- 7.2 ng of Dinophysistoxin-1 /g hepatopancreas (mean +/- SEM, N=6) were found in mussel extracts (Zone 8). These data showed that these shellfish samples are contaminated with the ester form 7-O-acyl-derivatives of Dinophysisyoxin-1, far beyond the safe regulatory limit. This paper also shows a direct relation between lipid content in the mussel tissue extracts and the levels of Dinophysistoxin-3. The 7-O-acyl-derivative dinophysistoxin-1 ester was the only compound associated with DSP toxins detected in the shellfish samples, and in view of the fact that metabolic transformation of Dinophysistoxin-3 into Dinophysistoxin-1 in humans has recently been described in the literature, the consumption of shellfish contaminated with 7-O-acyl-derivatives dinophysistoxin-1 could be a major reason that explains the diarrhetic symptoms shown by the intoxicated patients.

Metabolic transformation of dinophysistoxin-3 into dinophysistoxin-1 causes human intoxication by consumption of O-acyl-derivatives dinophysistoxins contaminated shellfish.

This paper describes for the first time a massive intoxication episode due to consumption of shellfish contaminated with 7-O-acyl-derivative dinophysistoxin-1, named Dinophysistoxin-3 (DTX-3). 7-O-acyl-derivative dinophysistoxin-1, a compound recently described in the literature, was found in shellfish samples collected in the Chilean Patagonia fjords. This compound does not inhibit Protein Phosphatases and also does not elicit the symptoms described for Diarrheic Shellfish Poisoning (DSP). The data showed here, give evidence of metabolic transformation of 7-O-acyl-derivative dinophysistoxin-1 (DTX-3) into Dinophysistoxin-1 (DTX-1, Methyl-Okadaic acid) in intoxicated patients. This metabolic transformation is responsible for the diarrheic symptoms and the intoxication syndrome showed by patients that consumed contaminated shellfish, which showed only the presence of 7-O-acyl-derivative dinophysistoxin-1. Patients fecal bacterial analysis for the presence of enteropathogens was negative and the mouse bioassay for DSP, performed as described for regulatory testing, was also negative. The HPLC-FLD and HPLC-MS analysis showed only the presence of DTX-3 as the only compound associated to DSP toxins in the contaminated shellfish samples. No other DSP toxins were found in the shellfish sample extracts. However, the patient fecal samples showed DTX-1 as the only DSP toxins detected in fecal. Moreover, the patient fecal samples did not show DTX-3. Since 7-O-acyl-derivative dinophysistoxin-1 (DTX-3) was the only compound associated to DSP toxins detected in the shellfish samples, an explanation for the diarrheic symptoms in the intoxicated patients would be the metabolic transformation of DTX-3 into DTX-1. This transformation should occur in the stomach of the poisoned patients after consuming 7-O-acyl-derivatives dinophysistoxin-1 (DTX-3) contaminated bivalves.

First evidence of Dinophysistoxin-1 ester and carcinogenic polycyclic aromatic hydrocarbons in smoked bivalves collected in the Patagonia fjords.

Diarrhetic shellfish poisoning (DSP) is a gastrointestinal disease caused by fat-soluble polyether toxins produced by dinoflagellates and accumulated in shellfish. Up to the present, only four fat-soluble polyethers have been known as diarrhetic shellfish toxins. Among them,*** Okadaic acid, Dinophysistoxin-1, Dinophysistoxin-2 and Dinophysistoxin-3. Outbreaks associated with DSP have occurred in the Chilean Patagonia fjords since 1970. Native people, who live in small communities close to the southern fjords, smoke fresh shellfish. During this popular smoking procedure, they impregnate the shellfish with polycyclic aromatic hydrocarbons, as incomplete combustion products, which are potent carcinogenic compounds, this product is sold in local markets without phycotoxins analysis or inclusion in any monitoring program. The present paper shows, DSP phycotoxins quantitation, using high performance liquid chromatography with fluorescent and mass spectrometric detection and the measurements of polycyclic aromatic hydrocarbons by gas chromatography with mass detection, in smoked shellfish samples. The presence of Dinophysistoxin-3, the Dinophysistoxin-1 ester (7-O-acyl-derivatives of dinophysistoxin-1), was assessed in all shellfish samples analyzed. The 7-OH in Dinophysistoxin-1 was esterified with palmitic fatty acid. The shellfish meat contains seven polycyclic aromatic hydrocarbons, among them fluoranthene, phenanthrene, anthracene, pyrene and benzo[a]pyrene. The gas chromatography-mass spectrometry analysis showed four of the six most frequent carcinogenic polycyclic aromatic hydrocarbons reported. The content of benzo[a]pyrene in the Razor Clam and Ribbed Mussel were 78.61 and 4.94 ng/g of shellfish dry weight, respectively. In both cases the benzo[a]pyrene amounts were greater than the acceptable tolerance limits of 1 microg/kg of sample. The Razor Clam samples also show amount further above the maximum label regulated by FAO/WHO (10 microg/kg). The presence of both type of compounds in the smoked shellfish samples analyzed, correspond to a dangerous combination, where the polycyclic aromatic hydrocarbons are carcinogenic compounds by themselves and DTX-1, is a potent tumor promoter.