Cyclic Peptide Hepatotoxins Research Articles

A 15-min non-competitive homogeneous assay for microcystin and nodularin based on time-resolved F\xf6rster resonance energy transfer (TR-FRET)

Simple and rapid methods are required for screening and analysis of water samples to detect cyanobacterial cyclic peptide hepatotoxins: microcystin/nodularin. Previously, we reported a highly sensitive non-competitive heterogeneous assay for microcystin/nodularin utilizing a generic anti-immunocomplex (anti-IC) single-chain fragment of antibody variable domains (scFv) isolated from a synthetic antibody library together with a generic adda ((2S,3S,4E,6E,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid)-specific monoclonal antibody (Mab) recognizing the common adda part of the microcystin/nodularin. Using the same antibody pair, here we report a homogeneous non-competitive assay for microcystin/nodularin based on TR-FRET (time-resolved Förster resonance energy transfer) measurement. The anti-IC scFv labeled with Alexa Fluor 680 and the Mab labeled with europium enabled the FRET process to occur in the presence of microcystin/nodularin. The TR-FRET signal is proportional to the toxin concentration in the sample. The rapid (15 min) homogeneous assay without requiring any washing step detected all the tested nine toxin variants (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF -LW, and nodularin-R). Very good signal to blank ratio (~13) was achieved using microcystin-LR and the sample detection limit (blank+3SD of blank) for microcystin-LR was ~0.3 μg/L (~0.08 μg/L in 80-μL reaction well). The practical application of the TR-FRET assay was demonstrated with water samples spiked with microcystin-LR as well as with environmental water. The average recoveries of microcystin-LR from spiked water ranged from 65 to 123%. Good correlation (r2 = 0.73 to 0.99) with other methods (liquid chromatography-mass spectrometry and previously reported heterogeneous assay) was found when environmental samples were analyzed. The developed wash-free assay has the potential to play as a quick screening tool to detect microcystin/nodularin from water below the World Health Organization’s guideline limit (1 μg/L of microcystin-LR).Graphical abstract

Mutagenesis of the Microcystin Tailoring and Transport Proteins in a Heterologous Cyanotoxin Expression System.

The microcystins are a large group of cyclic peptide hepatotoxins produced by several genera of freshwater cyanobacteria. The genes responsible for microcystin biosynthesis are encoded within a large (∼55 kbp) gene cluster, mcyA-J. The recent establishment of a cyanotoxin heterologous expression system in Escherichia coli has provided the means to study microcystin biosynthesis in a genetically tractable, rapidly growing host. Using this system, we demonstrate that deletion of the ABC-transporter, mcyH, and dehydrogenase, mcyI, abolishes microcystin production, while deletion of the O-methyltransferase, mcyJ, results in the production of the demethylated (DM) toxin [d-Asp3, DMAdda5]microcystin-LR. Both methylated and DM toxin variants were heterologously produced at high titers and efficiently exported into the extracellular medium, enabling easy purification. The results show that the mcy gene cluster can be engineered in E. coli to study the function of its individual components and direct the synthesis of particular microcystin variants. This technology could potentially be applied to other natural products of ecological and biomedical significance.

A fully automated system with on-line micro solid-phase extraction combined with capillary liquid chromatography–tandem mass spectrometry for high throughput analysis of microcystins and nodularin-R in tap water and lake water

Microcystins and nodularins are cyclic peptide hepatotoxins and tumour promoters from cyanobacteria. The present study describes the development, validation and practical application of a fully automated analytical method based on on-line micro solid-phase extraction–capillary liquid chromatography–tandem mass spectrometry for the simultaneous determination of seven microcystins and nodularin-R in tap water and lake water. Aliquots of just 100 μL of water samples are sufficient for the detection and quantification of all eight toxins. Selected reaction monitoring was used to obtain the highest sensitivity. Good linear calibrations were obtained for microcystins (50–2000 ng/L) and nodularin-R (25–1000 ng/L) in spiked tap water and lake water samples. Excellent interday and intraday repeatability were achieved for eight toxins with relative standard deviation less than 15.7% in three different concentrations. Acceptable recoveries were achieved in the three concentrations with both tap water matrix and lake water matrix and no significant matrix effect was found in tap water and lake water except for microcystin-RR. The limits of detection (signal to noise ratio = 3) of toxins were lower than 56.6 ng/L which is far below the 1 μg/L defined by the World Health Organization provisional guideline for microcystin-LR. Finally, this method was successfully applied to lake water samples from Tai lake and proved to be useful for water quality monitoring.

LC–ESI-Q-TOF-MS for faster and accurate determination of microcystins and nodularins in serum

Microcystins (MC) and nodularins (Nod) are cyclic peptide hepatotoxins and tumour promoters produced by cyanobacteria. This study deals with liquid chromatography–mass spectrometry (LC–MS) analyses of 9 major cyanobacterial peptide toxins, starting with a comparison of six small particle size reversed-phase HPLC columns, from which one, Phenomenex Synergi Hydro-RP, was chosen for further chromatography with accurate mass MS studies in a complex biological fluid, serum. The instrumentation used for the serum sample analysis included a Bruker micrO-TOF-Q-MS coupled to an Agilent 1200RR LC system. Total analysis run time per sample was 8.5 min. The Q-TOF-MS instrument was operated on auto MS–MS mode to obtain fragment ions (such as the characteristic fragment m/ z 135 from Adda amino acid residue) for toxin identification purposes. Detected mass errors in serum samples were in the range of from 0.3 mDa to 9.1 mDa. The narrow mass window (±20 mDa) for mass chromatograms used in quantitation gave benefits by background noise reduction. We conclude that a LC–ESI-Q-TOF-MS instrumentation is a powerful tool for identification and quantitation of cyanobacterial peptide toxins in a biological matrix.

ChemInform Abstract: Total Analysis System for Tumor Promoter Microcystins Produced by Cyanobacteria

Microcystins, produced by freshwater cyanobacteria, are cyclic peptide hepatotoxins and tumor promoters. An outbreak of human poisoning attributed to microcystins has been reported in Caruaru, Brazil in 1996, where exposure through renal dialysis led to the death of 50 patients. Although such severe acute effects on human health seem to be rare, microcystins poses problems to human health which could result from low-level, chronic exposure to microcystins in drinking water. It is therefore important to monitor the levels of microcystins in water reservoirs where cyanobacterial blooms occur. We have developed a total analysis system for microcystins using GC-MS and LC-MS. This comprises initial screening of samples to check for the presence of microcystins by detecting 2-methyl-3-methoxy-4-phenylbutyric acid as an ozonolysis product using thermospray interface LC-MS and electron ionization/GC-MS. If a sample is positive in a screening test, it will be necessary to follow through with identification and quantification. Frit-FAB interface LC-MS allowed the rapid identification of microcystins in cyanobacteria and lake water, and also enabled us to identify microcystins and their metabolites formed in vivo in mouse liver. Finally, Frit-FAB/LC-MS using selected ion monitoring could be used for quantitative analysis of microcystins in lake water in the low nanogram range. The total analysis system proposed in the present study should be applicable to studies of the metabolism of microcystins, of their detoxification, and those of the mechanism(s) of the accumulation in the food chain.

Chromatographic and capillary electrophoretic determination of microcystins

Microcystins are cyclic peptide hepatotoxins that are produced during blooming of cyanobacteria. In recent two decades they gained a great interest because of the related ecological and public health risks. Over 70 different analogs of microcystins have been isolated from natural blooms and laboratory cultures of cyanobacteria. Because they are inhibitors of protein phosphatases they act as tumor promoters, but on the other hand, inhibitive enzymatic and ELISA methods may be employed for their screening or quantitative determination. Much larger analytical potential for this purpose have, however high-performance separation chromatographic and electromigration techniques. Based on about 70 references from original journal papers these methods are reviewed together with presentation of their preconcentration methods and clean-up procedures for analyzing environmental samples. Some attention is also focused on increasing application of LC/MS methods, and comparison of separation techniques with immunochemical or enzymatic methods.

Determination of cyanobacterial cyclic peptide hepatotoxins in drinking water using CE

Four cyanobacteria hepatotoxins microcystin LR, microcystin RR, microcystin YR, and nodularin were simultaneously determined in drinking water using CZE and MEKC coupled with UV detection. The toxins were satisfactorily separated in both CZE and MEKC modes. Detection limits were in the range of 0.82-4.81 microg/mL, with R(2) values of 0.994-0.999. The linearity range tested for the standards was 5-100 microg/mL and RSD percentages were in the range of 1.0-2.5% for retention time and 3.0-10.2% for peak area. When a known amount of standard was spiked into a known volume of water and extracted, recoveries were 90.3% (RR), 101.5% (nodularin), 90.6% (YR), and 88.2% (LR). The use of SPE enabled cleanup and pre-concentration of a real sample to achieve a 100-fold concentration factor. Detection limits after SPE of the real sample spiked with microcystins were 0.090 microg/L (RR), 0.076 microg/L (YR), and 0.110 microg/L (LR), with RSD percentage values of 9.9-11.7% for peak area and 2.2-3.3% for retention time. The technique developed provides an alternative method for determining microcystins at levels of concentration that will be able to meet WHO drinking water guidelines for microcystins.

Development of an integrated laboratory system for the monitoring of cyanotoxins in surface and drinking waters

A system of analytical processes has been developed in order to serve as a cost-effective scheme for the monitoring of cyanobacterial toxins on a quantitative basis, in surface and drinking waters. Five cyclic peptide hepatotoxins, microcystin-LR, -RR, -YR, -LA and nodularin were chosen as the target compounds. Two different enzyme-linked immunosorbent assays (ELISA) were validated in order to serve as primary quantitative screening tools. Validation results showed that the ELISA methods are sufficiently specific and sensitive with limits of detection (LODs) around 0.1 μg/L, however, matrix effects should be considered, especially with surface water samples or bacterial mass methanolic extracts. A colorimetric protein phosphatase inhibition assay (PPIA) utilizing protein phosphatase 2A and p-nitrophenyl phosphate as substrate, was applied in microplate format in order to serve as a quantitative screening method for the detection of the toxic activity associated with cyclic peptide hepatotoxins, at concentration levels >0.2 μg/L of MC-LR equivalents. A fast HPLC/PDA method has been developed for the determination of microcystins, by using a short, 50 mm C18 column, with 1.8 μm particle size. Using this method a 10-fold reduction of sample run time was achieved and sufficient separation of microcystins was accomplished in less than 3 min. Finally, the analytical system includes an LC/MS/MS method that was developed for the determination of the 5 target compounds after SPE extraction. The method achieves extremely low limits of detection (<0.02 μg/L), in both surface and drinking waters and it is used for identification and verification purposes as well as for determinations at the ppt level. An analytical protocol that includes the above methods has been designed and validated through the analysis of a number of real samples.

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.

Molecular identification and evolution of the cyclic peptide hepatotoxins, microcystin and nodularin, synthetase genes in three orders of cyanobacteria

The cyanobacterial hepatotoxins, microcystin and nodularin, are produced by a wide range of cyanobacteria. Microcystin production has been reported in the four cyanobacterial orders: Oscillatoriales, Chroococcales, Stigonematales, and Nostocales. The production of nodularin is a distinct characteristic of the Nostocales genus Nodularia. A single rapid method is needed to reliably detect cyanobacteria that are potentially capable of producing these hepatotoxins. To this end, a PCR was designed to detect all potential microcystin and nodularin-producing cyanobacteria from laboratory cultures as well as in harmful algal blooms. The aminotransferase (AMT) domain, which is located on the modules mcyE and ndaF of the microcystin and nodularin synthetase enzyme complexes, respectively, was chosen as the target sequence because of its essential function in the synthesis of all microcystins as well as nodularins. Using the described PCR, it was possible to amplify a 472 bp PCR product from the AMT domains of all tested hepatotoxic species and bloom samples. Sequence data provided further insight into the evolution of the microcystin and nodularin synthetases through bioinformatic analyses of the AMT in microcystin and nodularin synthetases, with congruence between the evolution of 16S rRNA and the AMT domain.

Microcystins in Cyanobacterial Blooms from Two Freshwater Prawn (Macrobrachium rosenbergii) Ponds in Northern Thailand

The presence of cyanobacterially-produced microcystins (cyclic peptide hepatotoxins) was determined by analysis of Microcystis spp. in scum and water samples collected from a surface cyanobacterial bloom at a giant freshwater prawn (Macrobrachium rosenbergii) farm in Teung District, Chiang Rai Province, in northern Thailand during March to August 2004. M. aeruginasa and M. wesenbergii were the dominant species of cyanobacteria at concentrations between 850,000±190,000 and 302,000±73,000 colonies l -1 . Microcystins were present at 0.44±0.020 g kg -1 dry weight with microcystin-LR and microcystin-RR as the dominant microcystin types, accounting for 45% and 48% of the total microcystins detected, respectively. Microcystins in pond water were present at between 2.2±3.0 µg l-1 and 9.4±2.0 µg l -1 . Total microcystin concentrations in water seemed to be positively correlated with the number of Microcystis colonies. A decrease of microcystins in water was observed from April to August 2004, which may have resulted from removal by mechanisms not examined in this study. The total microcystin in water was slightly negatively correlated with total culturable bacteria numbers. Microcystis spp. colony number showed a significant negative correlation with soluble reactive phosphorus (r = -0.98, p<0.05). Nitrate-N, ammonium-N and soluble reactive phosphorus concentrations were between 1.2-1.9, 0.85-1.15 and 0.9-1.1 mg l-1 , respectively. Phosphorus concentrations were higher than the permitted limit for waste water from a fishery farm (less than 0.4 mg l-1 ). This study suggested that surface blooms of Microcystis species in cultivation ponds may present a risk for microcystin bioaccumulation in prawns, either directly or via other organisms in the food web.

Liquid chromatography–tandem mass spectrometry and accurate m/ z measurements of cyclic peptide cyanobacteria toxins

Microcystins are cyclic peptide hepatotoxins that are produced by several genera of cyanobacteria. We briefly review the molecular structural features of 67 reported cyclic heptapeptide microcystins and a related cyclic pentapeptide toxin. These substances present a significant analytical challenge because multiple toxins are often found in any given cyanobacteria or water sample, and it is likely that some structural variants have yet to be identified. We briefly describe a hybrid linear ion trap – Fourier transform ion cyclotron resonance (LT-FT-ICR) mass spectrometer equipped with a liquid chromatography (LC)/electrospray sample-introduction system. This instrument system was used to obtain LC/mass spectrometry (MS) data from the commercially available cyanobacteria toxins (microcystin-LR, -YR, -RR and nodularin). We review the electrospray mass spectra of these toxins and the significance of accurate m/ z measurements of toxin precursor and product ions. We discuss the collision-induced dissociation of [M + H] +, [M + H] 2+, and fragment ions in terms of ion-fragmentation pathways and mechanisms. The principal focus of this review is the potential for the efficient determination of microcystin structures with these MS techniques when no analytical standards are available or when new microcystin toxins are present in a sample.

Health Risk Assessment of Cyanobacterial (Blue-green Algal) Toxins in Drinking Water

Cyanobacterial toxins have caused human poisoning in the Americas, Europe and Australia. There is accumulating evidence that they are present in treated drinking water supplies when cyanobacterial blooms occur in source waters. With increased population pressure and depleted groundwater reserves, surface water is becoming more used as a raw water source, both from rivers and lakes/reservoirs. Additional nutrients in water which arise from sewage discharge, agricultural run-off or storm water result in overabundance of cyanobacteria, described as a 'water bloom'. The majority of cyanobacterial water-blooms are of toxic species, producing a diversity of toxins. The most important toxins presenting a risk to the human population are the neurotoxic alkaloids (anatoxins and paralytic shellfish poisons), the cyclic peptide hepatotoxins (microcystins) and the cytotoxic alkaloids (cylindrospermopsins). At the present time the only cyanobacteral toxin family that have been internationally assessed for health risk by the WHO are the microcystins, which cause acute liver injury and are active tumour promoters. Based on sub-chronic studies in rodents and pigs, a provisional Guideline Level for drinking water of 1 microg/L of microcystin-LR has been determined. This has been adopted in legislation in countries in Europe, South America and Australasia. This may be revised in the light of future teratogenicity, reproductive toxicity and carcinogenicity studies. The other cyanobacterial toxin which has been proposed for detailed health risk assessment is cylindrospermopsin, a cytotoxic compound which has marked genotoxicity, probable mutagenicity, and is a potential carcinogen. This toxin has caused human poisoning from drinking water, and occurs in water supplies in the USA, Europe, Asia, Australia and South America. An initial health risk assessment is presented with a proposed drinking water Guideline Level of 1 microg/L. There is a need for both increased monitoring data for toxins in drinking water and epidemiological studies on adverse health effects in exposed populations to clarify the extent of the health risk.

Bacterial Degradation of Microcystins and Nodularin

Microcystins and nodularins produced by cyanobacteria are potent hepatotoxins and tumor promoters. They are, respectively, cyclic heptapeptides and cyclic pentapeptides containing a characteristic beta-amino acid residue, (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E ),6(E)-dienoic acid (Adda). Strain B-9 isolated from Lake Tsukui, Japan, degrades microcystin-LR, which is the most toxic among the microcystins, to nontoxic Adda as an end product. In the present study, we characterized the bacterial degradation process of the cyclic peptide hepatotoxins by liquid chromatography/ion trap tandem mass spectrometry. The use of protease inhibitors with a B-9 cell extract indicated that the degradation process of microcystin-LR consists of sequential enzymatic hydrolyses of Arg-Adda, Ala-Leu, and then Adda-Glu peptide bonds into two known nontoxic intermediate degradation products and then Adda, respectively. Subsequently, additional microcystins and nodularin were compared with microcystin-LR on substrate specificity. The cyclic peptides containing the Arg-Adda peptide bond were almost completely degraded to Adda as well as microcystin-LR, whereas microcystin-LF containing the Phe-Adda peptide bond instead of Arg-Adda peptide bond and 6(Z)-Adda-microcystin-LR and -RR which are geometrical isomers of the Adda residue were barely degraded. These results indicated that the degrading enzymes selectively hydrolyzed the Arg-Adda peptide bond as the initial ring opening of the cyclic peptide hepatotoxins, microcystins and nodularin.

Derivatisation of microcystin with a redox-active label for high-performance liquid chromatography/electrochemical detection

Microcystins are a group of low molecular weight, cyclic peptide hepatotoxins. The most common detection and quantitation methods for these toxins are liquid chromatography with UV or mass spectrometric detections, phosphatase inhibition assays and enzyme-linked immunosorbent assays. In addition, derivatisation of these toxins with organic fluorophores followed by CE/laser induced fluorescence detection and HPLC/chemiluminescence detection; and with luminescent lanthanide chelates for competition assays have also been reported. However, the use of an electrochemical-active unit as a tag for microcystins has never been explored. Since the sulfhydryl group of 6-ferrocenylhexanethiol (Fc-C6-SH) can undergo a facile addition reaction with the α,β-unsaturated carbonyl group, this compound has been used as a redox-active labelling agent for a derivative of microcystins, microcystin-LR (MC-LR). The conjugate, Fc-MC-LR, has been isolated by high-performance liquid chromatography with electrochemical detection. The peak height-concentration curve was linear in the test range 20–400 ng of MC-LR (r value for linear regression >0.9987). The detection limit was determined to be ca. 18 ng MC-LR (S/N = 3). Meanwhile, the conjugate Fc-MC-LR has also been characterised by positive-ion electrospray-ionisation mass spectrometry. Electrochemical studies show that the adduct displays a reversible ferrocenium/ferrocene couple at ca. −0.040 V vs. SCE (scan rate = 50 mV s−1) in 0.1 M aqueous ammonium acetate–acetonitrile (55∶45 v/v).

Colorimetric Immuno-Protein Phosphatase Inhibition Assay for Specific Detection of Microcystins and Nodularins of Cyanobacteria

A novel immunoassay was developed for specific detection of cyanobacterial cyclic peptide hepatotoxins which inhibit protein phosphatases. Immunoassay methods currently used for microcystin and nodularin detection and analysis do not provide information on the toxicity of microcystin and/or nodularin variants. Furthermore, protein phosphatase inhibition-based assays for these toxins are not specific and respond to other environmental protein phosphatase inhibitors, such as okadaic acid, calyculin A, and tautomycin. We addressed the problem of specificity in the analysis of protein phosphatase inhibitors by combining immunoassay-based detection of the toxins with a colorimetric protein phosphatase inhibition system in a single assay, designated the colorimetric immuno-protein phosphatase inhibition assay (CIPPIA). Polyclonal antibodies against microcystin-LR were used in conjunction with protein phosphatase inhibition, which enabled seven purified microcystin variants (microcystin-LR, -D-Asp3-RR, -LA, -LF, -LY, -LW, and -YR) and nodularin to be distinguished from okadaic acid, calyculin A, and tautomycin. A range of microcystin- and nodularin-containing laboratory strains and environmental samples of cyanobacteria were assayed by CIPPIA, and the results showed good correlation (R2 = 0.94, P < 0.00001) with the results of high-performance liquid chromatography with diode array detection for toxin analysis. The CIPPIA procedure combines ease of use and detection of low concentrations with toxicity assessment and specificity for analysis of microcystins and nodularins.

Cytological alterations in isolated hepatocytes from common carp (Cyprinus carpio L.) exposed to microcystin-LR.

Microcystin-LR (MC-LR) is the most commonly encountered of the toxic cyclic peptide hepatotoxins occurring in China. It is a model compound for toxicological studies. In this study, the toxicity of MC-LR (50 and 500 micrograms/L) on isolated carp hepatocytes was determined and the ultrastructural alterations of cells induced by MC-LR were observed. The alterations noted when hepatocytes were exposed to 50 micrograms/L MC-LR were blebbing of cell membrane, shrinking and deformation of nuclei, vesiculation and transformation into concentric membrane whorls of RER, and swelling and rearrangement of the cytoskeleton. However, when the cells were exposed to 500 micrograms/L MC-LR, broken cell membranes, nuclei and cytoskeleton could be observed. These ultrastructural changes paralleled the pathological events, which lead to apoptosis or necrosis of hepatocytes. These results suggest that disruption of the cytoskeletal structures could account for the blebbing of cell membrane and apoptosis induced by MC-LR.

Total analysis system for tumor promoter microcystins produced by cyanobacteria

Microcystins, produced by freshwater cyanobacteria, are cyclic peptide hepatotoxins and tumor promoters. An outbreak of human poisoning attributed to microcystins has been reported in Caruaru, Brazil in 1996, where exposure through renal dialysis led to the death of 50 patients. Although such severe acute effects on human health seem to be rare, microcystins poses problems to human health which could result from low-level, chronic exposure to microcystins in drinking water. It is therefore important to monitor the levels of microcystins in water reservoirs where cyanobacterial blooms occur. We have developed a total analysis system for microcystins using GC-MS and LC-MS. This comprises initial screening of samples to check for the presence of microcystins by detecting 2-methyl-3-methoxy-4-phenylbutyric acid as an ozonolysis product using thermospray interface LC-MS and electron ionization/GC-MS. If a sample is positive in a screening test, it will be necessary to follow through with identification and quantification. Frit-FAB interface LC-MS allowed the rapid identification of microcystins in cyanobacteria and lake water, and also enabled us to identify microcystins and their metabolites formed in vivo in mouse liver. Finally, Frit-FAB/LC-MS using selected ion monitoring could be used for quantitative analysis of microcystins in lake water in the low nanogram range. The total analysis system proposed in the present study should be applicable to studies of the metabolism of microcystins, of their detoxification, and those of the mechanism(s) of the accumulation in the food chain.

Microcystin Uptake and Inhibition of Protein Phosphatases: Effects of Chemoprotectants and Self-Inhibition in Relation to Known Hepatic Transporters

The microcystins (Mcyst) are cyclic peptide hepatotoxins produced by cyanobacteria. They are chemically very stable and represent a public health threat when they occur in water bodies used for human consumption. Mice injected ip with Mcyst (LD50 50-100 μg/kg) accumulate Mcyst in the liver and die within 2-4 hr with massive intrahepatic hemorrhage. Pretreatment of mice with cyclosporin A (CP), rifamycin (Rif), trypan blue (TB), and trypan red (TR) protected the animals from a lethal dose of the toxin. The studies reported here using the freshly isolated rat hepatocyte model were undertaken in order (1) to evaluate the contribution of Mcyst transport in hepatocytes to the mechanism of chemoprotection for Mcyst in vivo toxicity by CP, Rif, TB, and TR and (2) to better characterize the hepatic Mcyst transporter in this model and determine its relationship to other bile acid/organic anion transporters that have already been fully described. Incubations with 125I-Mcyst were used to measure Mcyst uptake and accumulation in hepatocytes. It has been shown that at the cellular level Mcyst binds to and inhibits protein phosphatases 1 and 2A (PP) at nanomolar concentrations. Lethal doses of Mcyst in mice resulted in rapid profound inhibition of hepatic PP activity. PP activity was also inhibited in hepatocytes incubated with 100-500 nM Mcyst. PP inhibition in these studies was used as a marker of metabolic effects of the toxin. The chemoprotectants CP (5 μM), Rif(50 μM), TR (20 μM), and TB (20 μM) decreased accumulation of Mcyst (320 nM, a toxic concentration) after 30 min incubation to 37, 26, 30, and 66%, respectively, of that of cells treated with Mcyst only. Inhibition of PP activity in these cells was decreased. Inhibition of PP activity in hepatocytes was also decreased by known inhibitors of Mcyst transport: 50 μM of the bile acids cholate and taurocholate (TC) and 50 μM sulfobromophthalein. For all compounds tested the amount of Mcyst accumulated in the hepatocytes correlated qualitatively with the extent of PP inhibition. From these results it can be concluded that inhibition of Mcyst uptake by hepatocytes is the most likely mechanism of chemoprotection for Mcyst in vivo toxicity for TR, TB, CP, and Rif. Uptake of Mcyst was unaffected by changes in the ionic composition of the uptake buffer but was significantly decreased when PP activity of hepatocytes was inhibited by preincubation with Mcyst. This effect resulted directly from PP inhibition since preincubation with calyculin A (a cell permeant PP inhibitor, chemically unrelated to Mcyst) also led to inhibition of Mcyst uptake. In contrast, alanine and TC uptake were unaffected in cells with PP activity fully inhibited, indicating that the activities of these latter transporters, unlike that for Mcyst, are probably not controlled by serine/threonine phosphorylation. The pattern of inhibition, the lack of sodium or chloride dependency, and the sensitivity to PP inhibition indicate that the hepatic transport of Mcyst is unlikely to be via one of the already fully characterized sinusoidal bile salt organic anion transporters.

Use of a colorimetric protein phosphatase inhibition assay and enzyme linked immunosorbent assay for the study of microcystins and nodularins

Microcystins and nodularins are cyclic peptide hepatotoxins and tumor promoters produced by several genera of cyanobacteria. Using a rabbit anti-microcystin-LR polyclonal antibody preparation, the cross-reactivity with 18 microcystin and nodularin variants was tested. A hydrophobic amino acid, 3-amino-9-methoxy-10-phenyl-2,6,8-trimethyl-deca-4(E),6(E)-dienoic acid (Adda), which has the (E) form at the C-6 double bond in both microcystin and nodularin, was found essential for these toxins to express antibody specificity. Modification of -COOH in glutamic acid of microcystin and nodularin did not alter their antigenicity. Antibody cross-reactivity of these toxins was compared with their ability to inhibit protein phosphatase type 1 (PP1). Detection of PP1 inhibition was done by measuring the inhibition effect of the toxins on p-nitrophenol phosphate activity toward PP1. PP1 was obtained as recombinant PP1 expressed in E. coli. The inhibition effect of five microcystins and two nodularins on recombinant PP1 activity toward p-nitrophenol phosphate was measured in a microwell plate reader. The concentration of microcystin-LR causing 50% inhibition of recombinant PP1 activity (ic50) was about 0.3nM, while that of two modified microcystins had a significantly higher ic50. Microcystin-LR and nodularin with the (z) form of Adda at the C-6 double bond or having the monoester of glutamic acid did not inhibit PP1. These three toxins were also nontoxic in the mouse bioassay. These results show the importance of Adda and glutamic acid in toxicity of these cyclic peptides and that PPI inhibition is related to the toxins' mechanism of action.