The Role of Heterocysts in Cyanotoxin Production during Nitrogen Limitation.

Cyanobacteria harmful algal blooms (cyanoHABs) can have impacts on human health, aquatic ecosystems, and the economy. Nutrient management is an important mitigation and even remediation strategy. In this work, the paralytic shellfish toxin (PST)-producing Aphanizomenon (Aphan.) flos-aquae (Linnaeus) Ralfs ex Bornet & Flahault (now identified as Aphan. sp.) single filament isolate NH-5 was grown in P-depleted media, N-depleted media, and complete BG-11 media. Growth and heterocyst and vegetative cells were monitored using dry weight and cell counts. Ultrasonication was used to separate heterocysts from vegetative cells. HPLC-FLD with post-column derivatization was used to determine the saxitoxin (STX) and neosaxitoxin (NEOSTX) concentration per cell. Aphan. sp. NH-5 biomass was lower in the P-depleted media than in the N-depleted media and the control, though higher heterocyst counts were detected in the N-depleted media. The heterocyst toxin concentration was significantly higher compared to the vegetative cells for the N-depleted media, control, and P-depleted media. However, no significant differences were found among all preparations with regard to the STX-to-NEOSTX ratio. We conclude that N limitation induced higher heterocyst numbers and that N fixation activity is a factor behind the increase in the STX and NEOSTX production of Aphan. sp. NH-5.

Guanitoxin, re-naming a cyanobacterial organophosphate toxin.

Anatoxin-a(S) is the most potent natural neurotoxin produced by fresh water cyanobacteria. It is also the least understood and monitored. Although this potent cholinesterase inhibitor was first reported in the 1970s and connected with animal poisonings, the lack of chemical standards and identified biosynthetic genes together with limited diagnostics and acute reactivity of this naturally-occurring organophosphate have limited our understanding of its environmental breadth and human health implications. Anatoxin-a(S) irreversibly inhibits acetylcholinesterase much like other organophosphate agents like paraoxon. It is however often confused with the similarly named anatoxin-a that has a completely different chemical structure, mechanism of action, and biosynthesis. Herein we propose renaming of anatoxin-a(S) to clarify its distinct structure and mechanism and to draw renewed attention to this potent natural poison. We propose the new name guanitoxin (GNT) to emphasize its distinctive guanidino organophosphate chemical structure.

Re-evaluation of paralytic shellfish toxin profiles in cyanobacteria using hydrophilic interaction liquid chromatography-tandem mass spectrometry.

To date Paralytic shellfish toxin (PST) variants in cyanobacteria have primarily been characterized using high performance liquid chromatography coupled with fluorescence detection. In this study we re-evaluated the PST profiles of five cyanobacterial cultures (Dolichospermum circinale AWQC131C, Aphanizomenon sp. NH-5, Raphidiopsis raciborskii T3, Scytonema cf. crispum CAWBG524 and CAWBG72) and one environmental sample (Microseria wollei) using hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry. A total of 35 different PST variants were detected. D. circinale contained the highest number of variants (23), followed by S. cf. crispum CAWBG72 (21). Many of the variants detected in the cultures/environmental sample had not been reported from these strains previously: D. circinale (14 variants), S. cf. crispum CAWBG72 (16), S. cf. crispum CAWBG524 (9), Aphanizomenon sp. (9), R. raciborskii (7), and M. wollei (7). Of particular interest was the detection of M-toxins (Aphanizomenon sp., R. raciborskii, D. circinale). These have previously only been identified from shellfish where they were thought to be metabolites. Well-characterized PST variant profiles are essential for research investigating the genetic basis of PST production, and given that the toxicity of each variants differs, it will assist in refining risk assessments.

Health impacts from cyanobacteria harmful algae blooms: Implications for the North American Great Lakes.

Harmful cyanobacterial blooms (cHABs) have significant socioeconomic and ecological costs, which impact drinking water, fisheries, agriculture, tourism, real estate, water quality, food web resilience and habitats, and contribute to anoxia and fish kills. Many of these costs are well described, but in fact are largely unmeasured. Worldwide cHABs can produce toxins (cyanotoxins), which cause acute or chronic health effects in mammals (including humans) and other organisms. There are few attempts to characterize the full health-related effects other than acute incidences, which may go unrecorded. At present these are difficult to access and evaluate and may be ascribed to other causes. Such information is fundamental to measure the full costs of cHABs and inform the need for often-costly management and remediation. This paper synthesizes information on cHABs occurrence, toxicology and health effects, and relates this to past and current conditions in the Great Lakes, a major global resource which supplies 84% of the surface water in North America. This geographic region has seen a significant resurgence of cHABs since the 1980s. In particular we focus on Lake Erie, where increased reporting of cHABs has occurred from the early 1990's. We evaluate available information and case reports of cHAB-related illness and death and show that cHABs occur throughout the basin, with reports of animal illness and death, especially dogs and livestock. Lake Erie has consistently experienced cHABs and cyanotoxins in the last decade with probable cases of human illness, while the other Great Lakes show intermittent cHABs and toxins, but no confirmed reports on illness or toxicity. The dominant toxigenic cyanobacterium is the genus Microcystis known to produce microcystins. The presence of other cyanotoxins (anatoxin-a, paralytic shellfish toxins) implicates other toxigenic cyanobacteria such as Anabaena (Dolichospermum) and Lyngbya.

Sublethal microcystin exposure and biochemical outcomes among hemodialysis patients.

Cyanobacteria are commonly-occurring contaminants of surface waters worldwide. Microcystins, potent hepatotoxins, are among the best characterized cyanotoxins. During November, 2001, a group of 44 hemodialysis patients were exposed to microcystins via contaminated dialysate. Serum microcystin concentrations were quantified with enzyme-linked immunosorbent assay which measures free serum microcystin LR equivalents (ME). We describe serum ME concentrations and biochemical outcomes among a subset of patients during 8 weeks following exposure. Thirteen patients were included; 6 were males, patients' median age was 45 years (range 16-80), one was seropositive for hepatitis B surface antigen. The median serum ME concentration was 0.33 ng/mL (range: <0.16-0.96). One hundred thirty nine blood samples were collected following exposure. Patients' biochemical outcomes varied, but overall indicated a mixed liver injury. Linear regression evaluated each patient's weekly mean biochemical outcome with their maximum serum ME concentration; a measure of the extrinsic pathway of clotting function, prothrombin time, was negatively and significantly associated with serum ME concentrations. This group of exposed patients' biochemical outcomes display evidence of a mixed liver injury temporally associated with microcystin exposure. Interpretation of biochemical outcomes are complicated by the study population's underlying chronic disease status. It is clear that dialysis patients are a distinct 'at risk' group for cyanotoxin exposures due to direct intravenous exposure to dialysate prepared from surface drinking water supplies. Careful monitoring and treatment of water supplies used to prepare dialysate is required to prevent future cyanotoxin exposure events.

A putative gene cluster from a Lyngbya wollei bloom that encodes paralytic shellfish toxin biosynthesis.

Saxitoxin and its analogs cause the paralytic shellfish-poisoning syndrome, adversely affecting human health and coastal shellfish industries worldwide. Here we report the isolation, sequencing, annotation, and predicted pathway of the saxitoxin biosynthetic gene cluster in the cyanobacterium Lyngbya wollei. The gene cluster spans 36 kb and encodes enzymes for the biosynthesis and export of the toxins. The Lyngbya wollei saxitoxin gene cluster differs from previously identified saxitoxin clusters as it contains genes that are unique to this cluster, whereby the carbamoyltransferase is truncated and replaced by an acyltransferase, explaining the unique toxin profile presented by Lyngbya wollei. These findings will enable the creation of toxin probes, for water monitoring purposes, as well as proof-of-concept for the combinatorial biosynthesis of these natural occurring alkaloids for the production of novel, biologically active compounds.

A drinking water crisis in Lake Taihu, China: linkage to climatic variability and lake management.

In late May, 2007, a drinking water crisis took place in Wuxi, Jiangsu Province, China, following a massive bloom of the toxin producing cyanobacteria Microcystis spp. in Lake Taihu, China's third largest freshwater lake. Taihu was the city's sole water supply, leaving approximately two million people without drinking water for at least a week. This cyanobacterial bloom event began two months earlier than previously documented for Microcystis blooms in Taihu. This was attributed to an unusually warm spring. The prevailing wind direction during this period caused the bloom to accumulate at the shoreline near the intake of the water plant. Water was diverted from the nearby Yangtze River in an effort to flush the lake of the bloom. However, this management action was counterproductive, because it produced a current which transported the bloom into the intake, exacerbating the drinking water contamination problem. The severity of this microcystin toxin containing bloom and the ensuing drinking water crisis were attributable to excessive nutrient enrichment; however, a multi-annual warming trend extended the bloom period and amplified its severity, and this was made worse by unanticipated negative impacts of water management. Long-term management must therefore consider both the human and climatic factors controlling these blooms and their impacts on water supply in this and other large lakes threatened by accelerating eutrophication.

Occupational and environmental hazard assessments for the isolation, purification and toxicity testing of cyanobacterial toxins.

Cyanobacteria can produce groups of structurally and functionally unrelated but highly potent toxins. Cyanotoxins are used in multiple research endeavours, either for direct investigation of their toxicologic properties, or as functional analogues for various biochemical and physiological processes. This paper presents occupational safety guidelines and recommendations for personnel working in field, laboratory or industrial settings to produce and use purified cyanotoxins and toxic cyanobacteria, from bulk harvesting of bloom material, mass culture of laboratory isolates, through routine extraction, isolation and purification. Oral, inhalational, dermal and parenteral routes are all potential occupational exposure pathways during the various stages of cyanotoxin production and application. Investigation of toxicologic or pharmacologic properties using in vivo models may present specific risks if radiolabelled cyanotoxins are employed, and the potential for occupational exposure via the dermal route is heightened with the use of organic solvents as vehicles. Inter- and intra-national transport of living cyanobacteria for research purposes risks establishing feral microalgal populations, so disinfection of culture equipment and destruction of cells by autoclaving, incineration and/or chlorination is recommended in order to prevent viable cyanobacteria from escaping research or production facilities.

Microcystin analysis in human sera and liver from human fatalities in Caruaru, Brazil 1996.

In 1996, an extensive exposure of Brazilian hemodialysis patients at a dialysis center, using a municipal water supply water contaminated with cyanotoxins, provided the first evidence for acute lethal human poisoning from the cyclic peptide hepatotoxins called microcystins. During this outbreak, 100 of 131 patients developed acute liver failure and 52 of these victims were confirmed to have been exposed to lethal levels of microcystins. Detection and quantitation of microcystins in these biological samples posed some analytical challenges since there were no well-established and routine analytic methods to measure total microcystins in tissue or sera samples. At the time of the 1996 exposure we used analytic methods that combined the use of enzyme linked immunosorbant assay (ELISA), analytical high performance liquid chromatography (HPLC), electrospray ionization ion-trap mass spectroscopy (ES-ITMS) and matrix assisted laser desorption ionization-time of flight spectroscopy (MALDI-TOF). In the intervening years these methods have been improved and others developed that allow a more quantitative and critical analysis of microcystin contaminated tissue and sera. For these reasons, and to see how storage with time might effect the detection and stability of microcystins in these matrices, we reanalyzed selected liver tissues and sera from the Caruaru victims in Brazil. We developed and validated a procedure to measure total microcystins in Caruaru human sera and liver tissue using a combination of ELISA, liquid chromatography and liquid chromatography-mass spectrometry (LC/MS), GC/MS and MS/MS techniques. GC/MS and LC/MS were followed by MS/MS to obtain a fingerprint fragment spectra for the microcystins. The validity of the extraction procedure for free microcystins was confirmed by recovery experiments with blood sera spiked with microcystin-LR. We removed proteins with the Microcon Centrifugal Filter prior to LC/MS and ELISA analysis. A solid phase extraction (SPE) procedure was used for analysis of protein bound microcystins by conversion of ADDA to erythro-2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) combined with GC/MS. We found that the GC/MS method yielded a higher concentration of microcystin than that obtained by ELISA and LC/MS. We hypothesize that this difference is due to better GC/MS detection of the covalently bound form of microcystins in human liver tissue. We also concluded that microcystins are very stable when stored under these conditions for periods of almost 10 years.

Cyanobacteria toxins in the Salton Sea.

BACKGROUND: The Salton Sea (SS) is the largest inland body of water in California: surface area 980 km2, volume 7.3 million acre-feet, 58 km long, 14-22 km wide, maximum depth 15 m. Located in the southeastern Sonoran desert of California, it is 85 m below sea level at its lowest point. It was formed between 1905 and 1907 from heavy river flows of the Colorado River. Since its formation, it has attracted both people and wildlife, including flocks of migratory birds that have made the Salton Sea a critical stopover on the Pacific flyway. Over the past 15 years wintering populations of eared grebe (Podiceps nigricollis) at the Salton Sea, have experienced over 200,000 mortalities. The cause of these large die-offs remains unknown. The unique environmental conditions of the Salton Sea, including salinities from brackish freshwater at river inlets to hypersaline conditions, extreme daily summer temperatures (>38 degrees C), and high nutrient loading from rivers and agricultural drainage favor eutrophic conditions that encourage algal blooms throughout the year. A significant component of these algal blooms are the prokaryotic group - the Cyanophyta or blue-green algae (also called Cyanobacteria). Since many Cyanobacteria produce toxins (the cyanotoxins) it became important to evaluate their presence and to determine if they are a contributing factor in eared-grebe mortalities at the Salton Sea. RESULTS: From November 1999 to April 2001, 247 water and sediment samples were received for phytoplankton identification and cyanotoxin analyses. Immunoassay (ELISA) screening of these samples found that eighty five percent of all water samples contained low but detectable levels of the potent cyclic peptide liver toxin called microcystins. Isolation and identification of cyanobacteria isolates showed that the picoplanktonic Synechococcus and the benthic filamentous Oscillatoria were dominant. Both organisms were found to produce microcystins dominated by microcystin-LR and YR. A laboratory strain of Synechococcus was identified by PCR as being closest to known marine forms of this genus. Analyses of affected grebe livers found microcystins at levels that may account for some of the acute mortalities. CONCLUSION: The production of microcystins by a marine Synechococcus indicates that microcystins may be a more common occurrence in marine environments - a finding not recognized before this work. Further research should be done to define the distribution of microcystin producing marine cyanobacteria and to determine exposure/response effects of microcystins and possibly other cyanotoxins in the Salton Sea. Future efforts to reduce avian mortalities and remediate the Salton Sea should evaluate vectors by which microcystins enter avian species and ways to control and mitigate toxic cyanobacteria waterblooms at the Salton Sea.

LC/ESI/MS method development for the analysis of hepatotoxic cyclic peptide microcystins in animal tissues.

Microcystins (MCYSTs) are a family of related cyclic heptapeptides produced by several genera and species of blue-green algae (cyanobacteria). MCYSTs are potent and specific inhibitors of the serine threonine family of protein phosphatases, especially PP1 and PP2A. MCYSTs inhibit a liver's protein phosphatase by forming a covalent linkage between MCYSTs' Mdha residue and the phosphatase's cysteine residue. Due to the covalent linkage, analysis of MCYSTs in animal tissues has been limited to determination of unbound MCYST concentration. The MMPB (2-methyl-3-methoxy-4-phenylbutyric acid) oxidation procedure allows for the detection of total MCYST burden by releasing the carboxylic acid MMPB from MCYST's Adda amino acid. An internal standard 4-phenylbutyric acid (4PB) accounts for losses during the method. LC/MS conditions were developed using a ThermoFinnigan LCQDuo ion trap in negative electrospray ionization (ESI). Since both compounds produce the [M-H](-) ion, analysis occurs in selected ion monitoring (SIM) mode for both MMPB (m/z 207.1) and 4PB (m/z 163.1). Complete oxidation of MCYST-LR in liver tissues occurs in 3h. A solid phase extraction (SPE) cartridge removes MMPB and 4PB from the oxidant solution. The process efficiency for the SPE procedure is only 51.3%; however, suppression experiments indicate a 41.8% loss in signal strength due to matrix interferences. Therefore, the extraction efficiency for the SDB-XC cartridge procedure is 93.1%. This research has been successful in developing an LC/MS method for the analysis of total MCYST burden in animal tissues.

Sublethal exposure from microcystins to renal insufficiency patients in Rio de Janeiro, Brazil.

In November 2001, a cyanobacterial bloom dominated by Microcystis and Anabaena occurred in the Funil Reservoir and the Guandu River, both of which supply drinking water to Rio de Janeiro, Brazil. Using ELISA, microcystins were detected at a concentration of 0.4 microg/L in the drinking water, whereas a concentration of 0.32 microg/L was detected in activated carbon column-treated water for use at the renal dialysis center of Clementino Fraga Filho Hospital (HUCFF) at the Federal University of Rio de Janeiro. A total of 44 hemodialysis patients who received care at this center were believed to be exposed. Initial ELISA analyses confirmed the presence of serum microcystin concentrations > or = 0.16 ng/mL in 90% of serum samples collected from these patients. Twelve patients were selected for continued monitoring over the following 2-month period. Serum microcystin concentrations ranged from < 0.16 to 0.96 ng/mL during the 57 days after documented exposure. ELISA-positive samples were found throughout the monitoring period, with the highest values detected 1 month after initial exposure. ESI LC/MS analyses indicated microcystins in the serum; however, MS/MS fragmentation patterns typical of microcystins were not identified. LC/MS analyses of MMPB for control serum spiked with MCYST-LR. and patient sera revealed a peak at retention time of 8.4 min and a mass of 207 m/z. These peaks are equivalent to the peak observed in the MMPB standard analysis. Taken together ELISA, LC/MS, and MMPB results indicate that these renal dialysis patients were exposed to microcystins. This documents another incident of human microcystin exposure during hemodialysis treatment.

A simple colorimetric method to detect biological evidence of human exposure to microcystins.

Toxic cyanobacteria are contaminants of surface waters worldwide. Microcystins are some of the most commonly detected cyanotoxins. Biological evidence of human exposure may be difficult to obtain due to limitations associated with cost, laboratory capacity, analytic support, and expertise. We investigated the application of an enzyme-linked immunosorbant assay (ELISA) to detect microcystins in human serum. We analyzed ten serum samples collected from dialysis patients who were known to be exposed to a mixture of microcystins during a 1996 outbreak in Brazil. We applied a commercially available ELISA method to detect microcystins in serum, and we compared the ELISA results to a more specific method, liquid chromatography/mass spectrometry (LC/MS) that was also used to detect microcystins in serum. The Spearman correlation coefficient was calculated using serum microcystin concentrations in split samples obtained by the two methods. Serum microcystin concentrations were similar, and we found good correlation of microcystin concentrations between the two methods. The ELISA detected total microcystins, median=19.9 ng/ml; LC/MS detected microcystin-LR equivalents, median=21.2 ng/ml; Spearman r=0.96, p<0.0001. We found that ELISA is a simple, accessible method to screen human serum for evidence of microcystin exposure.

Cyanobacteria and prawn farming in northern New South Wales, Australia--a case study on cyanobacteria diversity and hepatotoxin bioaccumulation.

Harmful cyanobacteria pose a hazard to aquatic ecosystems due to toxins (hepatotoxic microcystins, nodularins, and cylindrospermopsin) they produce. The microcystins and nodularins are potent toxins, which are also tumor promoters. The microcystins and nodularins may accumulate into aquatic organisms and be transferred to higher trophic levels, and eventually affect vector animals and consumers. Prawn farming is a rapidly growing industry in Australia. Because information regarding effects of cyanobacteria at prawn farms was lacking, we examined diversity of cyanobacteria and toxin production plus bioaccumulation into black tiger prawns (Penaeus monodon) under both field (northern New South Wales, Australia, December 2001-April 2002) and laboratory conditions. Samples were analyzed for hepatotoxins using enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). The maximum density of cyanobacteria (1 x 10(6) to 4 x 10(6) cells/l) was reached in April. Cyanobacteria encountered were Oscillatoria sp. (up to 4 x 10(6) cells/l), Pseudanabaena sp. (up to 1.8 x 10(6) cells/l), Microcystis sp. (up to 3.5 x 10(4) cells/l), and Aphanocapsa sp. (up to 2 x 10(4) cells/l). An uncommon cyanobacterium, Romeria sp. (up to 2.2 x 10(6) cells/l), was also observed. Contrasting earlier indications, toxic Nodularia spumigena was absent. Despite that both Oscillatoria sp. and Microcystis sp. are potentially hepatotoxic, hepatotoxin levels in phytoplankton samples remained low (up to 0.5-1.2 mg/kg dw; ELISA) in 2001-2002. ELISA was found suitable not only for phytoplankton but prawn tissues as well. Enzymatic pretreatment improved extractability of hepatotoxin from cyanobacteria (nodularin from N. spumigena as an example), but did not generally increase toxin recovery from prawn hepatopancreas. There were slightly increasing hepatotoxin concentrations in prawn hepatopancreas (from 6-20 to 20-80 microg/kg dw; ELISA) during the study. Hepatotoxin concentrations in surface sediment remained low (<5 microg/kg dw; ELISA) throughout the study. Laboratory experiments indicated that prawn hepatopancreas, heart, and brain were primary organs for hepatotoxin bioaccumulation. Toxin concentration in other organs, including muscle, was less effective. Orally administered nodularin levels in hepatopancreas rapidly decreased from initial 830 to 250 microg/kg dw in 96 h. Similarly, concentration of microcystin-LR injected in prawns decreased from 130 to 30 microg/kg dw (hepatopancreas) in 2 h. These results demonstrate that potential risks caused by cyanobacteria in prawn farming (farmers, prawns, and consumers) were not substantial in 2001-2002. Although prawns may act as vectors for toxin transfer, they did not accumulate alerting amounts of hepatotoxins and were able to effectively detoxify them. Because bloom toxicity may vary, low-frequency toxin monitoring is recommended.

The uptake kinetics and immunotoxic effects of microcystin-LR in human and chicken peripheral blood lymphocytes in vitro.

Microcystin-LR is a cyanobacterial heptapeptide that presents acute and chronic hazards to animal and human health. We investigated the influence of this toxin on human and chicken immune system modulation in vitro. Peripheral blood lymphocytes were treated with microcystin-LR at environmentally relevant doses of 1, 10 and 25 microg/ml for 12, 24, 48, 72 h (for proliferation assay cells were treated for 72 h). T-cell and B-cell proliferation as well as apoptosis and necrosis were determined in human and chicken samples. IL-2 and IL-6 production by human lymphocytes also was measured. In addition, uptake kinetics of microcystin-LR into human and chicken peripheral blood lymphocytes were calculated by Liquid Chromatography (LS) /Mass Spectrometry (MS) analysis. At the highest dose microcystin-LR decreased T-cell proliferation and all doses of microcystin-LR inhibited B-cell proliferation. The frequency of apoptotic and necrotic cells increased in a dose and time-dependent manner. Human lymphocytes responded to stimulation with microcystin-LR by increased production of IL-6 and decreased production of IL-2. Human lymphocytes were able to uptake from 0.014 to 1.663 microg/ml and chicken lymphocytes from 0.035 to 1.733 microg/ml of the microcystin-LR added to the cultures, depending on the treatment time and dose. In conclusion, microcystin-LR acted as an immunomodulator in cytokine production and down-regulated lymphocyte functions by induction of apoptosis and necrosis. However, further studies dealing with the influence of microcystin-LR on expression cytokine genes and transcription factors are necessary to confirm these hypotheses.

Detection and analysis of the cyanobacterial peptide hepatotoxins microcystin and nodularin using SELDI-TOF mass spectrometry.

Surface-enhanced laser desorption ionization mass spectrometry (SELDI-TOFMS) was used to develop a new and useful method for determination and identification of the cyanobacterial (blue-green algae) toxins: microcystin and nodularin. The technique, combining chromatography and MS, enables microcystin/nodularin capture, purification, analysis, and processing from complex biological mixtures directly onto a hydrophobic chip. Factors affecting ion intensities, including matrix concentration and laser intensity, were investigated to optimize sensitivity of the method. Microcystins and nodularin were analyzed for femtomolar sensitivity (about 2.5 pg microcystin-LR in 2 microl water). Samples of blood sera and liver tissue were spiked with microcystin-LR and analyzed. The detection limit was 1 ng in 2 microl blood sera solution. Reactions of microcystins by compounds containing mercaptan groups, such as dithiothreitol, aminoethanethiol and protein phosphatase 1, were examined on the chip by mass spectrometry. Formation of the microcystin-dithiothreitol conjugate was used to confirm the target compounds. The MS/MS data obtained showed the presence of the microcystin conjugate. The reaction position of the toxin with target compound was confirmed by a series of MS/MS fragment ions. The protein profile of microcystins reacting with protein phosphatase 1 was also obtained from the SELDI-TOF mass spectra.

Estimation of microcystins in the freshwater fish Oreochromis niloticus in an Egyptian fish farm containing a Microcystis bloom.

Microcystins (MCYSTs) that accumulated in different organs of the freshwater fish Oreochromis niloticus, collected from a fish farm in Egypt containing heavy blooms of Microcystis aeruginosa, were investigated using an enzyme-linked immunosorbent assay (ELISA). The distribution of MCYSTs in the organs varied significantly. The highest MCYST level was recorded in the guts (821 ng/g fresh weight), followed by the livers (531.8 ng/g) and kidneys (400 ng/g). Smaller amounts of MCYST were detected in muscles (102 ng/g). The present study suggests that fish farms should be monitored for the presence of toxic cyanobacterial blooms to minimize the exposure of fish to potent hepatotoxins.

Human intoxication by microcystins during renal dialysis treatment in Caruaru-Brazil.

In February 1996, an outbreak of illness occurred at a hemodialysis clinic in Caruaru, Pernambuco State-Brazil. At this clinic 116 (89%) of 131 patients experienced visual disturbances, nausea, vomiting, and muscle weakness, following routine haemodialysis treatment. Subsequently, 100 patients developed acute liver failure. As of December 1996, 52 of the deaths could be attributed to a common syndrome now called 'Caruaru Syndrome'. Examination of previous years' phytoplankton counts showed that cyanobacteria were dominant in the water supply reservoir since 1990. Analyses of carbon and other resins from the clinic's water treatment system plus serum and liver tissue of patients led to the identification of two groups of hepatotoxic cyanotoxins: microcystins (cyclic heptapeptides) in all of these samples and cylindrospermopsin (alkaloid hepatotoxic) in the carbon and resins. Comparison of victims symptoms and pathology with animal studies on these two cyanotoxins, leads us to conclude that the major contributing factor to death of the dialysis patients was intravenous exposure to microcystins, specifically microcystin-YR, -LR and -AR. In 2000, a review of the Brazilian regulation for drinking water quality, promoted by Brazilian Health Ministry with collaboration of PAHO, incorporated cyanobacteria and cyanotoxins into this new regulation as parameters that must to be monitored for water quality control.

[ADMAdda5]-microcystins in Planktothrix agardhii strain PH-123 (cyanobacteria)--importance for monitoring of microcystins in the environment.

Two major and two minor microcystins (MCYST) were isolated from a hepatotoxic Danish strain of Planktothrix agardhii (Gomont) Anagnostidis et Komárek by reversed-phase high-performance liquid chromatography. The microcystins were characterized by UV spectroscopy, amino acid analysis, fast atom bombardment mass spectrometry (FABMS), and high-resolution FABMS. The major microcystins were further analysed by collisionally induced tandem electrospray ionization MS. The microcystins were found to be demethylated variants of MCYST-HtyR (homotyrosine-arginine) and MCYST-LR (leucine-arginine). The two major microcystins contained an acetyl-demethyl variant (ADMAdda) of 3-amino-9-acetoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda). This is the first report of [ADMAdda5]-microcystins in Planktothrix. The two [ADMAdda5]-microcystins inhibited protein phosphatase activity but showed low cross-reactivity with antibodies of an enzyme-linked immunosorbent assay (ELISA), emphasizing the potential underestimation of the toxicity of natural blooms dominated by Planktothrix when microcystin content is quantified using only an ELISA.