Production and specificity of mono and polyclonal antibodies against microcystins conjugated through N-methyldehydroalanine.

Microcystins (MCs) are a group of closely related toxic cyclic heptapeptides produced by common cyanobacteria (blue-green algae). Their toxicity is associated with specific inhibition of intracellular protein phosphatases type-1 and type-2A (PP1 and PP2A, respectively). We have developed a battery of antibodies to microcystins using chemical modification (aminoethylation) of one of its core amino acids, N-methyl-dehydroalanine. The developed antibodies displayed different reactivities to closely related MCs. Selected monoclonal antibodies were used for quantitative competitive ELISA assays. The analytical sensitivity of these assays was up to 1 ng/ml. Comparison of the developed ELISA tests with HPLC-based measurements of MCs in laboratory and field samples showed a good correspondence between the results yielded by these two methods. The antibodies developed by this technique provide the means for developing extremely sensitive and specific analytical assays for direct measurement of toxins in cyanobacterial or water samples.     

Measurement of oxidative stress parameters using liquid chromatography-tandem mass spectroscopy (LC-MS/MS)

There is increasingly intense scientific and clinical interest in oxidative stress and the many parameters used to quantify the degree of oxidative stress. However, there remain many analytical limitations to currently available assays for oxidative stress markers. Recent improvements in software, hardware, and instrumentation design have made liquid chromatography and tandem mass spectroscopy (LC-MS/MS) methods optimal choices for the determination of many oxidative stress markers. In particular, LC-MS/MS often provides the advantages of higher specificity, higher sensitivity, and the capacity to determine multiple analytes (e.g. 4-11 oxidative stress markers per LC run) when compared to other available methods, such as gas chromatography-MS, immunoassays, spectrophotometric or flourometric assays. LC-MS/MS methods are also compatible with cleanup and sample preparation methods including prior solid phase extraction or automated two dimensional LC/LC chromatography followed by MS/MS. LC-MS/MS provides three analytical filtering functions: (1) the LC column provides initial separation as each analyte elutes from the column. (2) The first MS dimension isolates ions of a particular mass-to-charge (m/z) ratio. (3) The selected precursor ion is fragmented into product ions that provide structural information about the precursor ion. Quantitation is achieved based on the abundances of the product ions. The sensitivity limits for LC-MS/MS usually lie within the range of fg-pg of analyte per LC on-column injection. In this article, the present capabilities of LC-MS/MS are briefly presented and some specific examples of the strengths of these LC-MS/MS assays are discussed. The selected examples include methods for isoprostanes, oxidized proteins and amino acids, and DNA biomarkers of oxidative stress.     

High grazer toxicity of [D-Asp,(E)-Dhb]microcystin-RR of Planktothrix rubescens as compared to different microcystins

Planktothrix rubescens, the dominant cyanobacterium in Lake Zürich, is generally considered to be toxic to zooplankton. The major toxin was determined by NMR spectroscopy and chemical analysis to be [D-Asp³,(E)-Dhb⁷]microcystin-RR. The compound was isolated in high purity, and its 24-h acute grazer toxicity was compared with microcystin-LR, microcystin-RR, microcystin-YR, and nodularin using a Thamnocephalus platyurus bioassay. Based on LC₅₀ values [D-Asp³,(E)-Dhb⁷]microcystin-RR was the most toxic microcystin tested. Nodularin was slightly more toxic under the conditions of the assay. The large number of individuals available for the grazer bioassay allowed the determination of dose-response curves of the different microcystins. These curves showed marked differences in their steepness. Microcystin-RR, which had nearly the same LC₅₀ as microcystin-LR and microcystin-YR, exhibited a very flat dose-response curve. This flat curve indicates that, for some individuals, lower concentrations of this microcystin are much more toxic than are the other two microcystins. Mortality of 100% requires much higher concentrations of microcystin-RR, indicating the resistance of some animals to the toxin. The purified [D-Asp³,(E)-Dhb⁷]microcystin-RR exhibited a higher molar absorption coefficient determined by quantitative amino acid analysis than the coefficients generally used for other microcystins. This observation has consequences for the risk assessment for microcystins and makes a structural determination of microcystins an absolute requirement. The presence of the dehydrobutyrine residue may be the reason for the higher specific toxicity of [D-Asp³,(E)-Dhb⁷]microcystin-RR when compared to the N-methyldehydroalanine-containing microcystins.     

Characterization of a microcystin and detection of microcystin synthetase genes from a Brazilian isolate of Nostoc

A nostocalean nitrogen-fixing cyanobacterium isolated from an eutrophic freshwater reservoir located in Piracicaba, São Paulo, Brazil, was evaluated for the production of hepatotoxic cyclic heptapeptides, microcystins. Morphologically this new cyanobacterium strain appears closest to Nostoc, however, in the phylogenetic analysis of 16S rRNA gene it falls into a highly stable cluster distantly only related to the typical Nostoc cluster. Extracts of Nostoc sp. CENA88 cultured cells, investigated using ELISA assay, gave positive results and the microcystin profile revealed by ESI-Q-TOF/MS/MS analysis confirmed the production of [Dha⁷]MCYST-YR. Further, Nostoc sp. CENA88 genomic DNA was analyzed by PCR for sequences of mcyD, mcyE and mcyG genes of microcystin synthetase (mcy) cluster. The result revealed the presence of mcyD, mcyE and mcyG genes with similarities to those from mcy of Nostoc sp. strains 152 and IO-102-I and other cyanobacterial genera. The phylogenetic tree based on concatenated McyG, McyD and McyE amino acids clustered the sequences according to cyanobacterial genera, with exception of the Nostoc sp. CENA88 sequence, which was placed in a clade distantly related from other Nostoc strains, as previously observed also in the 16S rRNA phylogenetic analysis. The present study describes for the first time a Brazilian Nostoc microcystin producer and also the occurrence of demethyl MCYST-YR variant in this genus. The sequenced Nostoc genes involved in the microcystin synthesis can contribute to a better understanding of the toxigenicity and evolution of this cyanotoxin.     

Simultaneous determination of amino acids in discrete brain areas in Suncus murinus by high performance liquid chromatography with electrochemical detection

An improved and simple reversed-phase high performance liquid chromatography method with electrochemical detection for the simultaneous determination of amino acids in brain tissue of Suncus murinus was developed. Homogenates from 5 different brain areas were derivatized with o-phthalaldehyde in the presence of sodium sulphite. Subsequent separation was achieved using linear gradient elution over 30 min. The derivatives were stable for up to 20 h at 4 °C. The method was accurate, reproducible, and showed good linearity. The recoveries were >88% for aspartate, glutamine, glutamate, glycine and γ-aminobutyric acid, with the limit of quantification varying from 5 to 30 pmol. The method was successfully applied for the measurement of amino acids under fed and fasted conditions.     

Detection of harmful cyanobacteria and their toxins by both PCR amplification and LC-MS during a bloom event.

We briefly report here the occurrence of toxic blooms in the eutrophic reservoir Billings, S?o Paulo city, Brazil. Water samples were collected in May 2004, during a cyanobacterial bloom. The presence of toxic species was confirmed by using PCR amplifications of a fragment region of genes encoding microcystin synthetase-mcyB. The determination of toxins was performed by liquid chromatography coupled with mass spectrometry (LC-MS). LC-MS analyses of the toxins from the bloom revealed variants of microcystins (MC), such as MC-LR, MC-RR and MC-YR. HPLC-FLD was used to determine the paralytic shellfish poisoning (PSP) saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 (GTX2) and 3 (GTX3). GTX2, GTX3 and NEO were detected for the first time in a natural sample from Billings reservoir. These results are a contribution to the knowledge of the biogeography of toxic cyanobacteria and their toxins, specifically in S?o Paulo.     

Microcystis Sp. Co-Producing Microcystin and Saxitoxin from Songkhla Lake Basin,Thailand

The Songkhla Lake Basin (SLB) located in Southern Thailand, has been increasingly polluted by urban and industrial wastewater, while the lake water has been intensively used. Here, we aimed to investigate cyanobacteria and cyanotoxins in the SLB. Ten cyanobacteria isolates were identified as Microcystis genus based on16S rDNA analysis. All isolates harbored microcystin genes, while five of them carried saxitoxin genes. On day 15 of culturing, the specific growth rate and Chl-a content were 0.2–0.3 per day and 4 µg/mL. The total extracellular polymeric substances (EPS) content was 0.37–0.49 µg/mL. The concentration of soluble EPS (sEPS) was 2 times higher than that of bound EPS (bEPS). The protein proportion in both sEPS and bEPS was higher than the carbohydrate proportion. The average of intracellular microcystins (IMCs) was 0.47 pg/cell on day 15 of culturing, while extracellular microcystins (EMCs) were undetectable. The IMCs were dramatically produced at the exponential phase, followed by EMCs release at the late exponential phase. On day 30, the total microcystins (MCs) production reached 2.67 pg/cell. Based on liquid chromatograph-quadrupole time-of-flight mass spectrometry, three new MCs variants were proposed. This study is the first report of both decarbamoylsaxitoxin (dcSTX) and new MCs congeners synthesized by Microcystis.     

Fates of Microcystis aeruginosa Cells and Associated Microcystins in Sediment and the Effect of Coagulation Process on Them

During toxic Microcystis aeruginosa blooms, large amounts of cells can enter sediment through natural settlement, and coagulation treatment used to control water blooms can enhance the accumulation of cells. However, the current understanding of the fates of these cells and associated microcystins (MCs), as well as the effect of coagulation treatment on these factors, is limited. The results of the present study show that Microcystis aeruginosa cells in sediment were steadily decomposed under experimental conditions, and that they completely disappeared within 28 days. The major MCs released from settled cells were immediately degraded in sediment, and microbial degradation may be the main mechanism involved in this process. Coagulation treatment with PAC (polyaluminium chloride) + sepiolite can efficiently remove Microcystis aeruginosa cells from the water column and prevent their re-invasion. Furthermore, coagulation treatment with PAC + sepiolite had no significant effect on the release and decomposition of MCs and, thus, will not enhance the MCs pollution. However, coagulation treatment can accelerate the nutrient cycle by enhancing the settlement of cells. More attention should be paid to the effect on nutrient cycle when coagulation treatment is used for restoration of aquatic ecosystems.     

The Effect of Cyanobacterial Biomass Enrichment by Centrifugation and GF/C Filtration on Subsequent Microcystin Measurement

Microcystins are cyclic peptides produced by multiple cyanobacterial genera. After accumulation in the liver of animals they inhibit eukaryotic serine/threonine protein phosphatases, causing liver disease or death. Accurate detection/quantification of microcystins is essential to ensure safe water resources and to enable research on this toxin. Previous methodological comparisons have focused on detection and extraction techniques, but have not investigated the commonly used biomass enrichment steps. These enrichment steps could modulate toxin production as recent studies have demonstrated that high cyanobacterial cell densities cause increased microcystin levels. In this study, three microcystin-producing strains were processed using no cell enrichment steps (by direct freezing at three temperatures) and with biomass enrichment (by centrifugation or GF/C filtration). After extraction, microcystins were analyzed using liquid chromatography-tandem mass spectrometry. All processing methods tested, except GF/C filtration, resulted in comparable microcystin quotas for all strains. The low yields observed for the filtration samples were caused by adsorption of arginine-containing microcystins to the GF/C filters. Whilst biomass enrichment did not affect microcystin metabolism over the time-frame of normal sample processing, problems associated with GF/C filtration were identified. The most widely applicable processing method was direct freezing of samples as it could be utilized in both field and laboratory environments.     

Standardization of microcystin extraction from fish tissues: A novel internal standard as a surrogate for polar and non-polar variants

Microcystins (MCs), a class of potent liver/hepatopancreatic toxins produced by numerous species of freshwater cyanobacteria, are well known for their toxic effects on aquatic organisms and humans. The extraction efficiencies of MCs can vary greatly as a result of matrix differences and/or differences in the extraction solvents, techniques, and clean-up steps utilized. Here we report the preparation of a unique internal standard, (S-hydroxypropyl-cys⁷)microcystin-LR (thiol-LR), with a mass different than any known MCs, which can be spiked into field samples and quantified via HPLC-MS along with endogenous MCs. Thiol-LR is modified at the Mdha residue, and therefore, provides an accurate measure of only free MCs that are not covalently bound to endogenous thiols in the matrix. The internal standard proved to be a good surrogate for MC-RR, -LR, and -LA in fish liver tissue. In fish muscle tissue, thiol-LR was a good surrogate for polar variants, MC-RR and -LR, but was less representative of the non-polar variant, MC-LA. Coupling of the internal standard (8 μg/g ww tissue) with HPLC-MS detection will standardize the quantification of free microcystins across species, tissue types, and extraction methods, making the estimation of exposure risk more reliable.     

Microcystins Alter Chemotactic Behavior in Caenorhabditis elegans by Selectively Targeting the AWA Sensory Neuron

Harmful algal blooms expose humans and animals to microcystins (MCs) through contaminated drinking water. While hepatotoxicity following acute exposure to MCs is well documented, neurotoxicity after sub-lethal exposure is poorly understood. We developed a novel statistical approach using a generalized linear model and the quasibinomial family to analyze neurotoxic effects in adult Caenorhabditis elegans exposed to MC-LR or MC-LF for 24 h. Selective effects of toxin exposure on AWA versus AWC sensory neuron function were determined using a chemotaxis assay. With a non-monotonic response MCs altered AWA but not AWC function, and MC-LF was more potent than MC-LR. To probe a potential role for protein phosphatases (PPs) in MC neurotoxicity, we evaluated the chemotactic response in worms exposed to the PP1 inhibitor tautomycin or the PP2A inhibitor okadaic acid for 24 h. Okadaic acid impaired both AWA and AWC function, while tautomycin had no effect on function of either neuronal cell type at the concentrations tested. These findings suggest that MCs alter the AWA neuron at concentrations that do not cause AWC toxicity via mechanisms other than PP inhibition.     

Analysis of amino acid enantiomers derived from antitumor antibiotics using chiral capillary electrophoresis

The chiral separation of enantiomeric forms of derivatized amino acids have been achieved based on a metal–chelate chiral capillary electrophoretic method and a cyclodextrin mediated host–guest interaction approach in micellar electrokinetic chromatography (MEKC) mode with laser-induced fluorescence detection. This approach has been applied to the determination of enantiomeric forms of amino acids derived from novel depsipeptide antitumor antibiotics, BMY-45012 and its analogs. Amino acids were analyzed by complete hydrolysis and the hydrolysate was derivatized with either dansyl chloride for UV absorbance detection or fluorescein isothiocyanate for laser based fluorescence detection. The presence of several amino acids, serine and β-hydroxyl-N-methy-valine in the proposed structure have been confirmed as d-serine and l-β-hydroxyl-N-methy-valine enantiomeric forms by both chiral capillary electrophoresis (chiral CE) and MEKC approaches. A non-chiral amino acid, sarcosine, was also confirmed. These methodologies provide a quick and sensitive approach for the determination of amino acids racemization of pharmaceutical natural products and have proven to be useful for structural elucidation refinement.     

Development of an in-line HPLC fingerprint ion-trap mass spectrometric method for identification and quality control of Radix Scrophulariae

Chromatographic fingerprinting has been widely accepted as a crucial method for qualitative and quantitative analyses of bioactives within traditional Chinese medicine. A fingerprint provides detailed information, specific for any given herb, thus facilitating the quality control measures of a given traditional Chinese medicine. In this article, quality assessment of Radix Scrophulariae was achieved by using high performance liquid chromatography combining diode-array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MS). Eight batches of sample obtained from different origins in China were used to establish the fingerprint and quantitative analyses. By comparing the retention times, UV and MS spectral data with reference standards, four characteristic peaks in the chromatograms were confirmed as corresponding to acetoside, angoroside C, cinnamic acid, and harpagoside. In addition, other two characteristic peaks were tentatively identified, following the literature interpretation of HPLC-ESI-MS and LC-MS/MS (affording structural information) to be sibirioside A and scrophuloside B₄, respectively. The results indicated that the newly developed HPLC-DAD-MS fingerprint method would be suitable for quality control of Radix Scrophulariae.     

Determination of steroidal glycosides in Yucca gloriosa flowers by LC/MS/MS

An high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) method, was developed for the quantitative analysis of the steroidal glycosides occurring in Yucca gloriosa flowers. The HPLC experiments were performed by means of an octadecyl-modified reversed-phase C-18 column and a binary mobile phase system under gradient elution conditions. The fragmentation patterns of steroidal saponins were analyzed by ESI–MSⁿ in positive ion mode and a specific multiple reaction monitoring MS/MS detection was developed for their quantitative determination. The described method provides high sensitivity and specificity for quantitative determination of the steroidal glycosides in Y. gloriosa flowers. Quantification was performed against an external calibration line obtained using each pure steroidal glycoside. Short- and long-term repeatabilities of the methods were better than 3 and 6%, respectively. The method was validated according to EMEA guidelines and applied to real samples.     

Comparison of the structure of key variants of microcystin to vasopressin

Microcystins (MCs) are cyclic heptapeptide compounds [where X(2) (position 2) and Z(4) (position 4) are variable l-aminoacids] produced by cyanobacteria and responsible for severe liver damage in animals ingesting acute doses of the toxic compounds. Certain variants of microcystins are more toxic than others, the differences being commonly ascribed to the hydrophobic nature of the variant. Microcystin-LR (MCLR) [X = l-leucine (L); Z = l-arginine (R); R1 = R2 = CH(3)] is the most toxic of all the microcystins investigated to date. This study investigates the similarity of the structures of MCLR and selected MC variants to the liver specific hormone vasopressin. Structures were compiled in HyperChem(?) (professional version 5.1). Initial comparisons of the MCLR and vasopressin indicated comparable volumes, surface areas and masses. Further studies using RMS overlays show that the microcystin derivative MCLR(Dha(7)) is comparably similar to vasopressin in terms of tertiary structure.     

Recent trends in development of biosensors for detection of microcystin

Increased cyanobacterial blooms, a source of cyanotoxins are linked with climate change and eutrophication in aquatic bodies, a major concern worldwide. Microcystins are potently hepatotoxic, nephrotoxic as well as carcinogenic. Thus microcystins are threat to tourism, agriculture and animal's health. However, there is a still lacuna in the knowledge of regulation of microcystins production. Presence of toxic and non-toxic cyanobacterial strains together and occurrence of various microcystin variants in aquatic bodies compounded the problem. Although several analytical techniques for microcystin detection such as bioassay, ELISA, HPLC and LC-MS etc. have been already prevalent, the development of biosensors offered rapid and accurate detection, high reproducibility and portability. Sequencing of Microcystis spp., opened the new vistas towards the development of biosensor at molecular and genetic level. This review incorporates the current trends in the development of biosensors for microcystin detection in the light of state-of-the-art techniques.     

The first study on the effects of microcystin-RR on gene expression profiles of antioxidant enzymes and heat shock protein-70 in Synechocystis sp. PCC6803

Microcystins are heptapeptide toxins produced by cyanobacteria. Microcystin-RR (MC-RR) is a common variant among the 80 variants identified so far. There have been many investigations documenting the toxic effects of microcystins on animals and higher plants, but little is known on the toxic effects of microcystins on algae, especially at molecular level. We studied the effects of MC-RR on gene expression profile of a few antioxidant enzymes and heat shock protein-70 (Hsp70) in Synechocystis sp. PCC6803. After two days post-exposure, a high dose toxin (5 mg/l, about 4.8 × 10⁻³ mM) significantly increased expression levels of the genes gpx1, sodB, katG, acnB, γ-TMT and dnaK2, while a relatively low dose toxin (1 mg/l, about 9.63 × 10⁻⁴ mM) induced a moderate and slow increase of gene expression. Our results indicate that MC-RR could induce the oxidative stress in Synechocystis sp. PCC6803 and the increase in gene expression of antioxidant enzymes and Hsp70 might protect the organism from the oxidative damage. In addition, cell aggregation was observed during the early period of exposure, which might be a specific oxidative stress reaction to MC-RR.     

Similar uptake profiles of microcystin-LR and -RR in an in vitro human intestinal model

Microcystins (MCs) are cyclic hepatotoxins produced by various species of cyanobacteria. Their structure includes two variable amino acids (AA) leading to more than 80 MC variants. In this study, we focused on the most common variant, microcystin-LR (MC-LR), and microcystin-RR (MC-RR), a variant differing by only one AA. Despite their structural similarity, MC-LR elicits higher liver toxicity than MC-RR partly due to a discrepancy in their uptake by hepatic organic anion transporters (OATP 1B1 and 1B3). However, even though ingestion is the major pathway of human exposure to MCs, intestinal absorption of MCs has been poorly addressed. Consequently, we investigated the cellular uptake of the two MC variants in the human intestinal cell line Caco-2 by immunolocalization using an anti-MC antibody. Caco-2 cells were treated for 30 min to 24 h with several concentrations (1-50 μM) of both variants. We first confirmed the localization of OATP 3A1 and 4A1 at the cell membrane of Caco-2 cells. Our study also revealed a rapid uptake of both variants in less than 1 h. The uptake profiles of the two variants did not differ in our immunostaining study neither with respect to concentration nor the time of exposure. Furthermore, we have demonstrated for the first time the nuclear localization of MC-RR and confirmed that of MC-LR. Finally, our results suggest a facilitated uptake and an active excretion of MC-LR and MC-RR in Caco-2 cells. Further investigation on the role of OATP 3A1 and 4A1 in MC uptake should be useful to clarify the mechanism of intestinal absorption of MCs and contribute in risk assessment of cyanotoxin exposure.     

A highly sensitive competitive enzyme immunoassay of broad specificity quantifying microcystins and nodularins in water samples

Microcystins (MCs) form a group of cyclic heptapeptides produced by common cyanobacteria (blue green algae) and cause both acute and chronic toxicity. For immunization purposes, an amino derivative of MC-LR was prepared before coupling to BSA. Among the different monoclonal antibodies produced, mAb MC159 was selected due to its broad specificity to develop a sensitive enzyme immunoassay (EIA). This method measures MC-LR, MC-YR, MC-LA, nodularins in a similar way and exhibits an important recognition (cross reactivity up to 69%) for Adda analogues. Using MC-LR as standard, the present EIA proved to be very sensitive with a limit of detection close to 10 fmol/ml, largely below the provisional guideline level for drinking water proposed by the WHO (1 pmol/ml for MC-LR). This assay showed a high accuracy (CV% < 12) and a high recovery rate for MC-LR in spiked surface water (up to 96.5%). Moreover due to its broad spectrum of recognition, this method allows a real quantification of the sum of MCs in water bloom and cyanobacteria culture samples. Indeed, in parallel analysis of these samples using HPLC, EIA shows a good relationship between both measurements while LC-MS/MS demonstrates the presence of different variants of MCs whose heterogeneity did not impair EIA measurement.