Synthesis and organotropism of 3H-dihydro derivatives of the cyanobacterial peptide hepatotoxin nodularin.

Tritium-labelled dihydro derivatives of the cyanobacterial peptide hepatotoxin nodularin were prepared by reduction with sodium boro[3H]hydride. The optimised reaction gave two dihydronodularin stereoisomers which were purified by high-performance liquid chromatography with a mobile phase of methanol-0.7% sodium sulfate (6:4) and a C(18) stationary phase. The specific activities of the stereoisomers were 1780-1807 dis min(-1) ng(-1). The radiolabelled dihydronodularins were tested for stability and used for toxicokinetic studies in mice. Liver was the main site of toxin accumulation.     

Transfer of nodularin to three-spined stickleback (Gasterosteus aculeatus L.), herring (Clupea harengus L.), and salmon (Salmo salar L.) in the northern Baltic Sea

Nodularin (NODLN) is a hepatotoxin produced by the cyanobacterium Nodularia spumigena, which occurs regularly in the Baltic Sea. The primary aim of this study was to study the transfer of NODLN to three-spined stickleback (Gasterosteus aculeatus L.), herring (Clupea harengus membras L.), and salmon (Salmo salar L.), which were caught from the northern Baltic Sea between August 2002 and August 2003. Liquid chromatography mass spectrometry (LC-MS) was used for NODLN analysis. NODLN was found in both herring (0–90 μg kg⁻¹ dw) and three-spined sticklebacks samples (2.8–700 μg kg⁻¹ dw). The recovery for the spiked stickleback samples in vitro was 28%. Only 1 salmon of a total of 10 contained a small amount of NODLN (10 μg kg⁻¹ dw). However, the high concentrations in individual stickleback suggest that possible transfer to higher trophic levels deserves more research.     

A new quantitative PCR assay for the detection of hepatotoxigenic cyanobacteria

Toxin-producing cyanobacteria are a worldwide threat to both human and animal health. The hepatotoxins microcystin and nodularin are the most commonly occurring toxins produced by bloom-forming cyanobacteria. They are cyclic peptides that are synthesized nonribosomally by a multienzyme complexes encoded within the microcystin (mcyS) and nodularin (ndaS) synthetase gene clusters. Early detection of potentially toxic blooms would allow for pre-emptive action to reduce consumer exposure to cyanotoxins. We have developed a quantitative PCR (qPCR) assay based on SYBR-green chemistry for the detection of potentially hepatotoxic cyanobacteria spanning all known microcystin and nodularin producing taxa using primers specifically targeting mcyE and ndaF. The qPCR assay was validated against previously analyzed cyanobacterial bloom samples. Whole cell qPCR using cultured M. aeruginosa PCC7806 and non-toxic M. aeruginosa UTEX2386 had a sensitivity of 1000 cells ml⁻¹. In summary, we have developed a robust and sensitive molecular method for the detection and quantification of hepatotoxigenic cyanobacteria in bloom samples. This technology offers several advantages over traditional and contemporary testing protocols currently used to assess water quality.     

Inhibition of protein phosphatases by microcystis and nodularin associated with hepatotoxicity

Summary Microcystins and nodularin, isolated from toxic blue-green algae, are hepatotoxic monocyclic polypeptides. Both microcystins and nodularin inhibited in vitro protein phosphatase activity present in a cytosolic fraction of mouse liver, bound to the okadaic acid receptors, protein phosphatases 1 and 2A, and thus resulted in the increase of phosphoproteins; this was referred to as the apparent activation of protein kinases. Their concentrations causing 50% of the maximal effects are comparable to that of okadaic acid, a potent protein phosphatase inhibitor and a potent tumor promoter, in the nanomolar range of concentration. The increase of phosphoproteins was observed in rat primary cultured hepatocytes and was subsequently associated with morphological changes, which appeared to be a step in the process of hepatotoxicity. The well-known hepatotoxic compounds,-amanitin and phalloidin, did not show any effects similar to those of microcystins, nodularin and okadaic acid. It is suggested that the hepatotoxicity of microcystins and nodularin may result from inhibition of protein phosphatases and the increase of phosphoproteins.     

State of the art on cyanotoxins in water and their behaviour towards chlorine

The occurrence of cyanobacterial blooms is drastically increasing in temperate countries and drinking water resources are threatened. As a result, cyanotoxins should be considered in water treatment to protect human health. This study presents a state of the art on cyanotoxins in water and their behaviour towards chlorination, a common drinking water disinfection process. Chlorination efficiency on cyanotoxins alteration depends on pH, chlorine dose and oxidant nature. Microcystins and cylindrospermopsin are efficiently transformed by chlorine, with respectively 6 and 2 by-products identified. In addition, chlorination of microcystins and cylindrospermopsin is associated with a loss of acute toxicity. Even though they have been less investigated, saxitoxins and nodularins are also altered by chlorine. For these toxins, no by-products have been identified, but the chlorinated mixture does not show acute toxicity. On the contrary, the fact that anatoxin-a has a very slow reaction kinetics suggests that this toxin resists chlorination.     

Extraction of 15 microcystins and nodularin using immunoaffinity columns.

Microcystins (MCYSTs) were isolated from surface water using reusable immunoaffinity columns. Individual MCYST were determined by high performance liquid chromatography equipped with a photo-diode array detector (HPLC-PDA, 200-300 nm). Subsequent analysis of the samples by liquid chromatography-electrospray ionization mass spectrometry (LC-ESMS) provided molecular weight information, which was used to tentatively identify individual MCYST variants for which standards were not available. Results obtained using immunoaffinity columns (IAC)-HPLC-PDA were compared to those obtained using solid phase extraction (SPE) Oasis HLB-HPLC-PDA. This is the first report of the extraction of 15 microcystins and nodularin using immunoaffinity columns. Whereas previous reports demonstrates the use of IAC for four microcystins, we found that IAC selectively extracted the following microcystins: MCYST-RR, [D-Asp3]MCYST-RR, MCYST-YR, MCYST-LR, 3 MCYST-LR variants, MCYST-AR, MCYST-FR, MCYST-WR, MCYST-LA, MCYST-LA variant, the less polar microcystins such as MCYST-LF, MCYST-LW and nodularin. The IAC extracts were free of interferences which enabled better detection and identification of MCYSTs. Based on the amount loaded to the cartridges, the method detection limit was 10-14 ng when using IAC and 25 ng for SPE of each MCYST-RR, MCYST-YR and MCYST-LR. Reproducibilities expressed as relative standard deviation were 6-10% for SPE and 4-17% for IAC.     

Detection of nodularin in flounders and cod from the Baltic Sea

The brackish water cyanobacterium Nodularia spumigena regularly forms waterblooms in the Baltic Sea. Many N. spumigena strains can produce nodularin, a hepatotoxic penta-peptide, which has caused several animal poisonings in the Baltic Sea area. To improve our understanding of nodularin bioaccumulation in aquatic organisms this study measured nodularin in flounder and cod caught from the Baltic Sea. Flounders were collected from the western Gulf of Finland in July 1996, September 1997, and September 1998, and from the Gulf of Bothnia in August 1997 and September 1998. Flounders were also collected from the coastal areas of Sweden in the Baltic Proper during September 1998. Cod were caught from the southern Baltic Sea in August 1998. Livers and muscles of the 1997 fish were isolated, extracted, and analysed for nodularin using high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) and protein phosphatase 1 (PP1) inhibition assay. Approximately 30-70 ng of nodularin/g dry weight (maximum value 140 ng/g) were found in the liver tissue samples by ELISA and PP1 inhibition. These concentrations were below the detection limit of HPLC. PP1 assay showed inhibition also in muscle samples, but this may due to other compounds present in the muscle extracts rather than NODLN or due to matrix interference. The recovery of nodularin from liver tissue with ELISA and PP1 assays was about 30%. Nodularin concentrations in samples are not corrected for recovery. Although the concentrations of nodularin found in this study are low further studies of nodularin are needed to assess possible bioaccumulation in brackish water food webs.     

Estimating the accumulation and transfer of Nodularia spumigena toxins by the blue mussel Mytilus edulis: an appraisal from culture and mesocosm experiments.

Accumulation of Nodularia spumigena toxins by Mytilus edulis was studied during laboratory and mesocosm experiments in order to investigate the possible pathways of nodularin in mussels and calculate toxin budgets. Mussels were exposed to 0.2-15.6 microg nodularin l(-1), fed for up to 5 days with Nodularia cells from culture, or blooming in different nutrient-treated seawater. Toxin concentration was monitored with LC-ESI-MS. During different exposures, the amount of nodularin detected in mussels increased linearly with increasing toxin concentration in food and attained 0.28-13.8 microg of nodularin g dw(-1) of the mussel whole body tissue after 12 h. The digestive gland was found to be the tissue with the highest toxin concentration. Nodularin concentration in faeces was not proportional to faeces production or to toxin concentration in food; however, it seemed to be mostly related to food quality as well as to food availability. The percentage of nodularin taken up by the mussels, relative to the amount contained in the offered food, varied from 10% to 20%, depending on food quality. During a 5-day toxin accumulation experiment, the acute reduction of the toxin in mussel tissues the second day and the following stabilization, showed that probably mussels maintain low toxin levels via efficient elimination and/or toxin metabolism. After a 72 h depuration period, mussels showed 75% reduction in their toxin content.     

Induction of Fas receptor and Fas ligand by nodularin is mediated by NF-κB in HepG2 cells

Nodularin is a natural toxin with multiple features, including inhibitor of protein phosphatases 1 and 2A as well as tumor initiator and promoter. One unique feature of nodularin is that this chemical is a hepatotoxin. It can accumulate into the liver after contact and lead to severe damage to hepatocyte, such as apoptosis. Fas receptor (Fas) and Fas ligand (FasL) system is a critical signaling network triggering apoptosis. In current study, we investigated whether nodularin can induce Fas and FasL expression in HepG2 cell, a well used in vitro model for the study of human hepatocytes. Our data showed nodularin induced Fas and FasL expression, at both mRNA and protein level, in a time- and dose-dependent manner. We also found nodularin induced apoptosis at the concentration and incubation time that Fas and FasL were significantly induced. Neutralizing antibody to FasL reduced nodularin-induced apoptosis. Further studies demonstrated that nodularin promoted nuclear translocation and activation of p65 subunit of NF-κB. By applying siRNA targeting p65, which knocked down p65 in HepG2 cells, we successfully impaired the activation of NF-κB by nodularin. In these p65 knockdown cells, we observed that Fas and FasL expression and apoptosis induced by nodularin were significantly reduced. These findings suggest the induction of Fas and FasL expression and thus cell apoptosis in HepG2 cells by nodularin is mediated through NF-κB pathway.     

Nucleotide excision repair impairment by nodularin in CHO cell lines due to ERCC1/XPF inactivation

The problem of toxicity of cyanobacterial toxins is of increasing concern, as the incidence of such blooms grows. Among the toxins, the most abundant in the environment are hepatotoxins known as nodularins and microcystins. These toxins are responsible for almost all known cases of fresh and brackish water intoxication and are responsible for recurrent episodes of human and animal illness and death. Moreover, they are believed to be potent tumor promoters and initiators. However, the mechanisms by which these toxins induce liver cancer are not well understood. The aim of the present study was to determine the effect of nodularin on the kinetics of nucleotide excision repair (NER) in Chinese hamster ovary (CHO) cells exposed to UV radiation. The first set of experiments was performed to define the optimal treatment conditions for nodularin to avoid the possibility of encountering false positive signals in the comet assay due to the apoptogenic activity of nodularin. Based on the analysis of apoptosis, the 6-h treatment time of cells with nodularin (1mug/ml, 10mug/ml and 20mug/ml) was chosen for the alkaline comet assay. The kinetics of NER was determined in CHO cell lines: AA8 (wild-type) and mutant cell lines: UV135 (XPG(-)), UV41 (XPF(-)) and UV20 (ERCC1(-)) exposed to 20J/m(2) UV radiation. The micronucleus assay was performed to determine a residual DNA damage in four cell lines treated with nodularin (10mug/ml) and exposed to equitoxic doses UV radiation. Radiation doses of UV producing 50% of survival for AA8, UV135, UV20 and UV41 cell lines were calculated from UV survival curves. The results show that nodularin impairs the incision/excision step of NER in CHO cells by the ERCC1/XPF inactivation and leads to an increased level of UV-induced cytogenetic DNA damage.