Diversity of hepatotoxic microcystins and bioactive anabaenopeptins in cyanobacterial blooms from Greek freshwaters (original) (raw)
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Detection and monitoring toxigenicity of cyanobacteria by application of molecular methods
Environmental Toxicology, 2006
The aim of this study was early genetic identification of microcystin-producing cyanobacteria and monitoring their toxigenicity by determining toxin concentrations in three Polish lakes throughout the summer of 2004. The assessment of cyanobacterial blooms was carried out in shallow, eutrophic water bodies: Lake Jeziorak, Lake Bninskie, and Sulejow Reservoir. Samples for DNA, phycological, and toxin analyses were collected from July till October. Molecular analysis of the 16S rRNA region was used to detect cyanobacteria in water samples. The microscopic analysis was performed to investigate seasonal variation of phytoplankton. Cyanobacteria, with domination by Microcystis, Planktothrix, and Planktolyngbya were detected during the whole monitoring period in Sulejow Reservoir, Lake Bninskie, and Lake Jeziorak, respectively. The presence and identification of toxic strains in water bodies was studied by PCR amplification of mcy genes in the microcystis synthesis pathway. The presence of the mcyA, mcyB, mcyD, and mcyE genes in water samples indicated the genetic potential to produce microcystins. Toxicity of water samples and microcystin concentrations were established by PPIA and HPLC, respectively. The maximum concentration of microcystins was 11.13 μg/L and 4.67 μg/L in samples dominated by P. agardhii and M. aeruginosa, respectively. Molecular analysis showed that toxigenic strains of cyanobacteria occurred in the three lakes throughout the summer season. © 2006 Wiley Periodicals, Inc. Environ Toxicol 21: 380–387, 2006.
Environmental Toxicology, 2009
Toxin-producing cyanobacteria in lakes and reservoirs form a threat to humans as well as various forms of aquatic life. This study is an investigation into the occurrence and distribution of Microcystins (MCYST) in 13 Greek Lakes. The distribution of MCYST in water and surface scum and toxin bioaccumulations in the omnivorous fish species Carassius gibelio were surveyed in all lakes. Considerable amounts of MCYST were found in water and scum of all lakes, irrespective of the trophic state, the type of the lake, and the reported dominant cyanobacterial species. Toxin accumulation in six tissues (liver, brain, intestine, kidney, ovary, and muscle) of C. gibelio was also analyzed. Even though the target organ for MCYST is the liver, in our study, MCYST were found also in the rest of C. gibelio tissues in the following order: liver > intestine > kidney > brain > ovaries > muscle. Risk assessments were carried out, taking into account the WHO guidelines and the tolerable daily intake (TDI) for MCYST. Our findings suggest that the amounts of MCYST found in water of Lakes Kastoria, Koronia, Pamvotis, Doirani, Mikri Prespa, Petron, and Zazari, pose adverse health risks. Also, it is likely to be unsafe to consume C. gibelio in Lakes Koronia, Kastoria, Pamvotis, and Mikri Prespa due to the high concentrations of accumulated MCYST. © 2009 Wiley Periodicals, Inc. Environ Toxicol 25: 418–427, 2010.
Environmental Toxicology, 2009
Tai Lake is the third largest freshwater lake in China with annual cyanobacteria blooms. Microcystins produced by these blooms have serious health risks for populations surrounding the lake, especially for people living on Tai Lake, because they usually drink raw lake water after a simple alum treatment. This study presents data on the detection and identification of microcystins in waters used for daily life by people living on Tai Lake, during the cyanobacterial blooming in July 2007. The health risks from drinking these microcystin-polluted waters were also calculated. The main microcystins detected by high-performance liquid chromatography-electrospray ionization mass spectrometry in the water samples collected from two parts of Tai Lake (Wuli Lake and Meiliang Bay) were MC-LR (4.33-12.27 lg/L), MC-RR (8.36-16.91 lg/L) and MC-YR (1.41-5.57 lg/L). Risk assessment showed that the drinking water simply treated by alum was not safe. The lowest calculated hazards ratios in all water samples was 6.4, which indicated that the risk of microcystins exposure from drinking water was over six times higher than the tolerable daily intake (TDI) recommended by The World Health Organization (WHO). Further studies should be conducted to elucidate the relationships between the epidemiology of people living on Tai Lake and microcystins exposure from drinking water. # 2008 Wiley Periodicals, Inc. Environ Toxicol 24: 82-86, 2009.
Archives of Environmental Contamination and Toxicology, 2006
Toxic cyanobacterial blooms are a worldwide problem, causing serious water pollution and public health hazard to humans and livestock. The intact cells as well as the toxins released after cellular lysis can be responsible for toxic effects in both animals and humans and are actually associated with fish kills. Two fish cell lines-PLHC-1 derived from a hepatocellular carcinoma of the topminnow Poeciliopsis lucida and RTG-2 fibroblast-like cells derived from the gonads of rainbow trout Oncorhynchus mykiss were exposed to several concentrations of extracts from a natural cyanobacterial bloom and a Microcystis aeruginosa-isolated strain. After 24 hours, morphologic and biochemical changes (total protein content, lactate dehydrogenase leakage, neutral red uptake, methathiazole tetrazolium salt metabolization, lysosomal function, and succinate dehydrogenase [SDH] activity) were investigated. The most sensitive end point for both cyanobacterial extracts in PLHC-1 cells was SDH activity, with similar EC 50 values (6 lM for the cyanobacterial bloom and 7 lM for the isolated strain). RTG-2 cells were less susceptible according to SDH activity, with their most sensitive end point lysosomal function with an EC 50 of 4 lM for the M. aeruginosa-isolated strain and 72 lM for the cyanobacterial bloom. The lysosomal function was stimulated at low concentrations, although SDH activity increased at high doses, indicating lysosomal and energetic alterations. Increased secretion vesicles, rounding effects, decreased cell numbers and size, hydropic degeneration, esteatosis, and apoptosis were observed in the morphologic study. Similar sensitivity to the M. aeruginosa-isolated strain was observed in both cell lines, whereas the cyanobacterial bloom was more toxic to the PLHC-1 cell line. A common consequence of the eutrophication of inland waters is the overgrowth of cyanobacteria (blue-green algae) causing ''cyanobacterial blooms.'' These cyanobacteria can produce toxins and release them to the water as a consequence of their lysis, thus becoming an important water-quality problem in many countries where toxic cyanobacterial blooms have been reported (Moreno et al. 2003a; Nasri et al. 2004). Microcystins (MCs) are the most commonly found group of cyanotoxins. These hepatotoxins are produced by several genera of cyanobacteria, including Microcystis, Nodularia, Oscillatoria, Anabaena, and Nostoc spp. (Carmichael 1994). MCs are heptapeptides with a basic cyclic structure (D-Ala-L-X-erythro-b-mehtyl-D-iso-Asp-L-Y-Adda-D-iso-Glu-N-methylde-hydro-Ala). The residue, Adda, a characteristic cyanobacterial b-amino acid, refers to 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid, and X and Y represent the variable amino-acid residues in the cyclic structure (Dawson 1998). MCs are known to affect many organisms from microalgae to mammals (Figueiredo et al. 2004; Montagnolli et al. 2004) and are associated with fish kills (Carbis et al. 1997; Zimba et al. 2001). Moreover, freshwater fish as well as other aquatic organisms could not only be damaged by cyanobacterial toxins but are also able to bioaccumulate them (Magalhaes et al. 2003; Mohamed et al. 2003), thus making the ingestion of contaminated food a human health risk. The toxicity of cyanobacterial blooms has been investigated traditionally by exposing rodents to MCs from extracts of these blooms and then monitoring their survival, behavior, and histopathology (Teneva et al. 2002). Aune and Berg (1986) were among the first investigators to suggest the use of isolated rat hepatocytes for quick and inexpensive investigation of the toxicity of cyanobacterial blooms. Recently, this model system was shown to correlate particularly well with the content of MCs (Heinze et al. 2001). Nevertheless, inasmuch as the hepatocytes assay still requires the killing of animals, screening cyanobacterial extracts for their impact on cells might be more easily performed using established cell lines (Teneva et al. 2002). The use of cell lines has become particularly attractive in recent years because of the development of techniques applicable to multiwell plate readers and because of the increased availability of cell cultures originating from nonmammalian animals, such as fish, which allow cyanobacterial extracts to be evaluated with respect to environmental
Toxins produced in cyanobacterial water blooms - toxicity and risks
Interdisciplinary Toxicology, 2009
Cyanobacterial blooms in freshwaters represent a major ecological and human health problem worldwide. This paper briefly summarizes information on major cyanobacterial toxins (hepatotoxins, neurotoxins etc.) with special attention to microcystins -cyclic heptapeptides with high acute and chronic toxicities. Besides discussion of human health risks, microcystin ecotoxicology and consequent ecological risks are also highlighted. Although significant research attention has been paid to microcystins, cyanobacteria produce a wide range of currently unknown toxins, which will require research attention. Further research should also address possible additive, synergistic or antagonistic effects among different classes of cyanobacterial metabolites, as well as interactions with other toxic stressors such as metals or persistent organic pollutants.
Occurrence of the cyanobacterial toxin cylindrospermopsin in northeast Germany
Environmental Toxicology, 2007
The frequent occurrence of the cyanobacterial toxin cylindrospermopsin (CYN) in the (sub)tropics has been largely associated with cyanobacteria of the order Nostocales of tropical origin, in particular Cylindrospermopsis raciborskii. C. raciborskii is currently observed to spread northwards into temperate climatic zones. In addition, further cyanobacteria of the order Nostocales typically inhabiting water bodies in temperate regions are being identified as CYN-producers. Therefore, data on the distribution of CYN in temperate regions are necessary for a first assessment of potential risks due to CYN in water used for drinking and recreation. A total of 127 lakes situated in the north-eastern part of Germany were investigated in 2004 for the presence of the toxin CYN and the phytoplankton composition. The toxin could be detected in half of the lakes (n ¼ 63) and in half of 165 samples (n ¼ 88). Concentrations reached up to 73.2 g CYN/g DW. CYN thus proved more widely distributed than previously demonstrated. The analyses of phytoplankton data suggest Aphanizomenon sp. and Anabaena sp. as important CYN producers in Germany, and confirm recent findings of Aphanizomenon flos-aquae as CYN-producing species frequently inhabiting water bodies in temperate climatic regions. The data shown here suggest that CYN may be an important cyanobacterial toxin in German water bodies and that further data are needed to assess this. # 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 26-32, 2007.
Occurrence and diversity of cyanotoxins in Greek lakes
Scientific Reports
Toxic cyanobacteria occur in Greek surface water bodies. However, studies on the occurrence of cyanotoxins (CTs) are often limited to mainly microcystins (MCs), with use of screening methods, such as ELISA, that are not conclusive of the chemical structure of the CT variants and can be subject to false positive results. A multi-lake survey in Greece (14 lakes) was conducted in water and biomass, targeted to a wide range of multi-class CTs including MCs, nodularin-R (NOD), cylindrospermopsin (CYN), anatoxin-a (ANA-a) and saxitoxins (STXs), using multi-class/variant LC-MS/MS analytical workflows, achieving sensitive detection, definitive identification and accurate quantitation. A wide variety of CTs (CYN, ANA-a, STX, neoSTX, dmMC-RR, MC-RR, MC-YR, MC-HtyR, dm 3 MC-LR, MC-LR, MC-HilR, MC-WR, MC-LA, MC-LY, MC-LW and MC-LF), were detected, with MCs being the most commonly occurring. In biomass, MC-RR was the most abundant toxin, reaching 754 ng mg −1 dw, followed by MC-LR (458 ng mg −1 dw). CYN and ANA-a were detected for the first time in the biomass of Greek lakes at low concentrations and STXs in lakes Trichonis, Vistonis and Petron. The abundance and diversity of CTs were also evaluated in relation to recreational health risks, in a case study with a proven history of MCs (Lake Kastoria). Cyanobacteria are photosynthetic prokaryotic organisms, which can rapidly multiply, forming "blooms" in water 1,2. They are known to produce various metabolites of diverse and mostly unknown function 3,4 as well as potent toxins, called cyanotoxins (CTs) 5-8. CTs are compounds with diverse structures and biosynthetic origin (alkaloid, heterocyclic, peptide, aminoacids, etc) 6,7,9,10 with various modes of toxicity (e.g. hepatotoxic, dermatotoxic, neurotoxic, cytotoxic) 11,12. They are therefore, potentially harmful to humans and other organisms 13-16 , posing a significant ecological risk to aquatic habitats and to public health 2,17,18. Microcystins (MCs) are the most widespread class of CTs detected in fresh waters 9. They are cyclic heptapeptides (Fig. S1) containing the unusual β-amino acid ADDA ((2 S,3 S,8 S,9 S)−3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl deca-4,6-dienoic acid) which is responsible for their toxicity due to its conjugated diene, the cyclic structure and the non-esterified Glu residue 19-21. MCs are known to be hepatotoxic. They mainly mediate their toxicity by uptake into hepatocytes, followed by inhibition of protein phosphatases (PPs), which are able to dephosphorylate serine and threonine residues. Inhibition of PPs results in an increased phosphorylation of proteins in liver cells, significantly affecting metabolic pathways, membrane transport, secretion, etc. 19. At sub-lethal doses MCs are also known to be potent liver tumor promoters 22 and produce oxidative DNA damage 23,24. Up to now, over 248 MC variants have been identified and structurally characterized 25,26. Nodularins (NODs) are pentapeptides with similar structure with MCs. Ten variants have been identified so far, among which Nodularin-R (NOD) is the most frequently found 25,27,28. Cylindrospermopsin (CYN) is an alkaloid cyanotoxin (Fig. S1) of rising environmental concern, due to its multiple toxicity endpoints, frequency of occurrence and severity of health impacts 29,30. CYN is mainly hepatotoxic, but potential effects also include genotoxicity, dermatotoxicity, fetal toxicity and cytotoxicity 29-31. Unlike other CTs (MCs, NODs) that are mostly intracellular in viable cells, CYN is found mostly as extracellular 32. Anatoxin-a (ANA-a) is a secondary, bicyclic amine alkaloid (Fig. S1), which is highly water-soluble, presenting increased neurotoxicity 33,34. The (+) entantiomer, which is toxicologically active, has been associated with a number of animal fatalities, including cattle, dogs, bats, pigeons and flamingos 35-37. Saxitoxins (STXs), also known as Paralytic Shellfish Poisoning toxins (PSPs), are relatively
Environmental Toxicology, 2001
In an aquatic ecosystem, during cyanobacterial bloom lysis, a mixture of toxins and other cyanobacterial and bacterial components will be present in the water, acting on aquatic organisms. Most of the research into toxic effects of cyanobacteria has involved the use of purified toxins. In this study, the "real-life" situation of a cyanobacterial lysis event was investigated. For this purpose, intact cells from a natural cyanobacterial bloom from Lake Müggelsee, Berlin, were taken and the cells were broken by repeated freeze/thaw cycles. This crude extract was used to expose several aquatic organisms ranging from microalgae (Scenedesmus armatus), macrophyte (Ceratophyllum demersum), invertebrate (Chaoborus crystallinus) up to fish eggs (Danio rerio) to look at several physiological parameters such as detoxication enzyme activity and, in the case of the microalgae and the macrophyte, also the effect on activity of photosynthesis. In all the tests, the cyanobacterial crude extract caused stronger effects than the pure cyanobacterial toxins used in equivalent concentrations.
Cyanotoxins: Bioaccumulation and Effects on Aquatic Animals
Marine Drugs, 2011
Cyanobacteria are photosynthetic prokaryotes with wide geographic distribution that can produce secondary metabolites named cyanotoxins. These toxins can be classified into three main types according to their mechanism of action in vertebrates: hepatotoxins, dermatotoxins and neurotoxins. Many studies on the effects of cyanobacteria and their toxins over a wide range of aquatic organisms, including invertebrates and vertebrates, have reported acute effects (e.g., reduction in survivorship, feeding inhibition, paralysis), chronic effects (e.g., reduction in growth and fecundity), biochemical alterations (e.g., activity of phosphatases, GST, AChE, proteases), and behavioral alterations. Research has also focused on the potential for bioaccumulation and transferring of these toxins through the food chain. Although the herbivorous zooplankton is hypothesized as the main target of cyanotoxins, there is not unquestionable evidence of the deleterious effects of cyanobacteria and their toxins on these organisms. Also, the low toxin burden in secondary consumers points towards biodilution of microcystins in the food web as the predominant process. In this broad review we discuss important issues on bioaccumulation and the effects of cyanotoxins, with emphasis on microcystins, as well as drawbacks and future needs in this field of research.