Cyanobacterial 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

A homogeneous assay for rapid detection of cyanobacterial peptide hepatotoxins: Microcystins and nodularins

A homogeneous assay for rapid detection of cyanobacterial peptide hepatotoxins: Microcystins and nodularins

Comparative Cellular Toxicity of Hydrophilic and Hydrophobic Microcystins on Caco-2 Cells

Microcystins (MC), cyanobacterial peptide hepatotoxins, comprise more than 100 different variants. They are rather polar molecules but some variants contain hydrophobic amino acid residues in the highly variable parts of the molecule. In MC-LF and MC-LW, the more hydrophobic phenylalanine (F) and tryptophan (W), respectively, have replaced arginine (R) in MC-LR. Depending on the structure, microcystins are expected to have different in vivo toxicity and bioavailability, but only a few studies have considered the toxic properties of the more hydrophobic variants. The present study shows that MC-LF and MC-LW have more pronounced cytotoxic effects on Caco-2 cells as compared to those of MC-LR. Treatment of Caco-2 cells with MC-LW and especially MC-LF showed clear apoptotic features including shrinkage and blebbing, and the cell–cell adhesion was lost. An obvious reduction of cell proliferation and viability, assessed as the activity of mitochondrial dehydrogenases, was observed with MC-LF, followed by MC-LW and MC-LR. Cytotoxicity was quantified by measuring lactate dehydrogenase leakage. The more hydrophobic MC-LW and MC-LF induced markedly enhanced lactate dehydrogenase leakage compared to controls and MC-LR, indicating that the plasma membrane was damaged. All of the three toxins examined inhibited protein phosphatase 1, with MC-LF and MC-LW to a weaker extent compared to MC-LR. The higher toxic potential of the more hydrophobic microcystins could not be explained by the biophysical experiments performed. Taken together, our data show that the more hydrophobic microcystin variants induce higher toxicity in Caco-2 cells.

Fast separation of microcystins and nodularins on narrow-bore reversed-phase columns coupled to a conventional HPLC system

Six modern narrow-bore (50 mm length × 2–2.1 mm internal diameter) reversed-phase HPLC columns were tested in the rapid separation of ten microcystins and three nodularins, cyanobacterial peptide hepatotoxins. The columns had either a sub-3 μm particle size or were of monolithic silica technology for high efficiency and rapid run times. A standard low-pressure gradient HPLC equipment with diode-array UV detector was used for the chromatography. The gradient mobile phase consisted of water and acetonitrile, both acidified with trifluoroacetic acid, and the gradient rise times were 1–4 min. Chromatographic performance was assessed by visual judgement and by calculating parameters such as capacity factors, resolution, peak width, selectivity and peak form. Several chromatographic conditions produced excellent results. We recommend sub-3 μm particle-based or monolithic reversed-phase columns for routine use in microcystin and nodularin HPLC analyses.

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 μl water). Samples of blood sera and liver tissue were spiked with microcystin-LR and analyzed. The detection limit was 1 ng in 2 μl 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.

First observation of microcystin-LR in pelagic cyanobacterial blooms in the northern Baltic Sea

Cyanobacterial bloom samples from the Gulf of Finland (northern Baltic Sea) were collected in July 2003 and analyzed for microcystins and nodularins, cyanobacterial peptide hepatotoxins, by ELISA, HPLC-UV and LC-MS. The blooms consisted mainly of the genera Nodularia, Anabaena and Aphanizomenon. The main hepatotoxin in the samples was nodularin-R (Nod-R), all the samples also contained demethylnodularin-R. The presence of microcystin-LR was confirmed in three locations out of nine by multiple reactant monitoring on the triple quadrupole mass spectrometer. This is the first reported finding of microcystins in the Baltic Sea from the open sea area. Anabaena was the likely producer of microcystin-LR in the samples.

High-Throughput Screening of Ten Microcystins and Nodularins, Cyanobacterial Peptide Hepatotoxins, by Reversed-Phase Liquid Chromatography-Electrospray Ionisation Mass Spectrometry

Liquid chromatography-electrospray ionisation mass spectrometry was evaluated in the high-throughput analysis of microcystins and nodularins, cyanobacterial peptide hepatotoxins. Extracts originating from cyanobacterial strains and field material were separated on a 30 mm × 4 mm I.D. Merck Purospher STAR RP-18e column using a rapid gradient of aqueous formic acid and acetonitrile, ionised by electrospray technique and analysed on a Micromass Quattro II triple-quadrupole mass spectrometer operated in the selected ion recording (SIR) mode. The total analysis time per sample was 2.8 min corresponding to 514 samples a day. The system showed good robustness during a series of 320 repetitive injections of a field sample containing three major microcystins.

Screening for cyanobacterial hepatotoxins, microcystins and nodularin in environmental water samples by reversed-phase liquid chromatography–electrospray ionisation mass spectrometry

Water samples taken from 93 freshwater and brackish water locations in Åland (SW Finland) in 2001 were analysed for biomass-bound microcystins and nodularin, cyanobacterial peptide hepatotoxins, by liquid chromatography–mass spectrometry (LC–MS) in selected ion recording (SIR) and multiple reaction monitoring modes, HPLC–UV, and enzyme-linked immunosorbent assay (ELISA). The extracted toxins were separated on a short C 18 column with a gradient of acetonitrile and 0.5% formic acid, and quantified on a Micromass Quattro Micro triple-quadrupole mass spectrometer with an electrospray ion source operated in the positive SIR or scan mode. An injection of 50 pg of microcystin-LR, m/z 995.5, on column gave a signal-to-noise ratio of 17 (peak-to-peak) at the chosen SIR conditions. In-source or MS–MS fragmentation to m/z 135.1, a fragment common to most microcystins and nodularin, was used for confirmatory purposes. Microcystins with a total toxin concentration equal to or higher than 0.2 μg l −1 were confirmed by all three methods in water samples from 14 locations. The highest toxin concentration in a water sample was 42 μg l −1. The most common toxins found were microcystins RR, LR and YR with different degrees of demethylation (non-, mono- or didemethylated). Parallel results achieved with ELISA and HPLC–UV were generally in good agreement with the LC–MS SIR results.

A colorimetric and fluorometric microplate assay for the detection of microcystin-LR in drinking water without preconcentration

Protein phosphatase inhibition assays currently used for the detection of cyanobacterial peptide hepatotoxins in drinking water require an enrichment step using C18 cartridges to achieve lower the detection limit. This paper describes a colorimetric and fluorometric protein phosphatase inhibition method for the direct detection of microcystin-LR (MCYST-LR) in drinking water without complex clean-up steps and preconcentration procedures. In this assay three different substrates, p-nitrophenyl phosphate (p-NPP) and two fluorogenic compounds, 4-methylumbelliferyl phosphate (MUP) and 6,8-difluoro-4-methylumbelliferyl phosphate DiFMUP), were tested. The detection limits of the assay are 0.25 and 0.1 μg/l using colorimetric and fluorometric methods, respectively. These levels are well below the provisional guideline value for MCYST-LR of 1 μg/l of drinking water. The detection limit of the fluorometric method is comparable to that of the classical ELISA test. Although both the latter tests allow the detection of MCYST-LR in drinking water directly without pretreatment, the protein phosphatase inhibition assay remain less expensive and therefore more attractive for use in the routine assessment of drinking water contamination by microcystins.

Determination of cyanobacterial hepatotoxins directly in water using a protein phosphatase inhibition assay

A colorimetric phosphatase inhibition assay using protein phosphatase 2A and p-nitrophenyl phosphate as substrate for determining cyanobacterial peptide hepatotoxins directly in water without sample preconcentration has been developed. The assay uses commercially available materials and is much more simple to use than similar procedures using radiolabelled substrates. It has similar sensitivity to the radiolabelled assays and, with a working range of around 0.2–1 μg/L, is able to determine these toxins at concentrations below the provisional World Health Organisation guideline of 1 μg/L for microcystin-LR. The method appears robust and not to be affected by the sample matrix apart from possibly some components of cellular material if present at very high levels in extracts of cyanobacterial material. It is not affected by the presence of low levels of methanol in sample extracts.

A time-resolved fluoroimmunometric assay for the detection of microcystins, cyanobacterial peptide hepatotoxins

An immunoassay based on the time-resolved fluorometry (TR-FIA) was developed for microcystins, cyanobacterial peptide hepatotoxins. The assay was performed in a competitive mode and it utilised the monoclonal antibodies raised against microcystin-LR, and a europium chelate of microcystin-LR as a competitive antigen. The sensitivity of the assay was 0.1 μg/l. The detection method of TR-FIA was compared to a commercially available kit based on the enzyme-linked immunosorbent assay (ELISA). The same level of sensitivity could be obtained with TR-FIA (in a non-optimised system). The simplified method of TR-FIA leads to a shorter analysis time.

Isolation and detection of microcystins and nodularins, cyanobacterial peptide hepatotoxins.

Some species and strains within the freshwater cyanobacterial (blue-green algal) genera Microcystis, Oscillatoria (also known as Planktothrix), Anabaena, and Nostoc are known to produce cyclic heptapeptide liver toxins, microcystins. Closely related toxic cyclic pent ape ptides, nodularins, have been isolated from the brackish water cyanobacterium Nodularia spumigena. In addition, some cyanobacteria produce neurotoxic compounds such as anatoxin-a, anatoxin-a(s), and saxitoxins. Recent reviews of the chemical and biological properties of microcystins, nodularins, and other cyanobacterial toxins are given in refe. (, , , ).

Electrochemical detection of microcystins, cyanobacterial peptide hepatotoxins, following high-performance liquid chromatography

A novel amperometric HPLC detection method for the cyanobacterial (blue–green algal) peptide toxins microcystin-LR, -YR and -RR was developed. Purified microcystins and cyanobacterial extracts were chromatographed using an internal surface reversed-phase column with acetate- and phosphate-based mobile phase systems. Electrochemical oxidation reactions at 1.20 V vs. Ag/AgCl (glassy carbon working electrode) were shown to originate in arginine and tyrosine residues of microcystins.

Choosing analytical strategy for microcystins

This review evaluates methods for detection and quantification of microcystins, cyanobacterial peptide hepatotoxins. The assessed methods include bioassays. high-performance liquid chromatography o...

Screening for cyanobacterial toxins in bloom and strain samples by thin layer chromatography

Two thin layer chromatographic (TLC) procedures were evaluated for the capacity screening of cyanobacterial peptide hepatotoxins and anatoxin-a in laboratory-cultured strain samples and in natural bloom samples. Seventy-five samples previously investigated with the reference methods, the chief of which was mouse bioassay, were analyzed blindly by TLC. Forty-three of the samples were hepatotoxic according to the reference methods, and 39 of them were found to contain peptide hepatotoxins by TLC coupled to u.v.-densitometry. Seven samples contained anatoxin-a according to the reference methods, and five of these were found to contain anatoxin-a by TLC, using derivatization with Fast Black K salt. Four of the samples which were neurotoxic by mouse bioassay also contained hepatotoxins by TLC. Twenty-four samples were nontoxic according to the reference methods, and in 23 of these no toxins were found by TLC either.

Destruction of cyanobacterial peptide hepatotoxins by chlorine and chloramine

Toxic cyanobacteria (blue-green algae) are becoming recognized as a greater water quality problem as blooms become more common in waters affected by increasing nutrient levels, and knowledge of the properties of the toxin increases. Of particular importance are the hepatotoxic cyclic peptides produced by certain strains of Microcystis species and by Nodularia spumigena. The effects of chlorine and chloramine on these hepatotoxins in both freeze-dried and intact material were investigated. The destruction of the toxins was monitored using HPLC analysis. In a number of cases mouse bioassays were used for confirmation of the destruction of the toxins and to ensure that toxic by-products, not detectable by HPLC, were not formed. Contrary to evidence reported in the literature, chlorine was effective in destroying toxins, as long as sufficient chlorine was used. Toxins were destroyed under conditions such that a chlorine residual of at least 0.5 mg/l was present after 30 min contact time. The destruction of toxins was pH dependent; chlorinating agents such as calcium and sodium hypochlorite were not as effective at high dose rates due to elevation of pH. Chloramination had little effect on toxins. Although the degradation mechanisms have yet to be established, the results show that chlorination may be an effective and practical method for the removal of cyanobacterial peptide toxins from drinking water.