Investigation of microcystin conformation and binding towards PPP1 by molecular dynamics simulation.

Microcystins (MC) are a group of structurally similar cyanotoxins with currently 279 described structural variants. Human exposure is frequent by consumption of contaminated water, food or food supplements. MC can result in serious intoxications, commensurate with ensuing pathology in various organs or in rare cases even mortality. The current WHO risk assessment primarily considers MC-LR, while all other structural variants are treated as equivalent to MC-LR, despite that current data strongly suggest that MC-LR is not the most toxic MC, and toxicity can be very different for MC congeners. To investigate and analyse binding and conformation of different MC congeners, we applied for the first time Molecular Dynamics (MD) simulation to four MC congeners (MC-LR, MC-LF, [Enantio-Adda5]MC-LF, [ß-D-Asp3,Dhb7]MC-RR). We could show that ser/thr protein phosphatase 1 is stable in all MD simulations and that MC-LR backbone adopts to a second conformation in solvent MD simulation, which was previously unknown. We could also show that MC congeners can adopt to different backbone conformation when simulated in solvent or in complex with ser/thr protein phosphatase 1 and differ in their binding behaviour. Our findings suggest that MD Simulation of different MC congeners aid in understanding structural differences and binding of this group of structurally similar cyanotoxins.

Erratum to Machine learning prediction of cyanobacterial toxin (microcystin) toxicodynamics in humans.

In this manuscript, which appeared in ALTEX (2020), 37(1), 24-36, doi:10.14573/altex.1904031 , there were errors in Tables 1 and 3.

Machine learning prediction of cyanobacterial toxin (microcystin) toxicodynamics in humans.

Microcystins (MC) represent a family of cyclic peptides with approx. 250 congeners presumed harmful to human health due to their ability to inhibit ser/thr-proteinphosphatases (PPP), albeit all hazard and risk assessments (RA) are based on data of one MC-congener (MC-LR) only. MC congener structural diversity is a challenge for the risk assessment of these toxins, especially as several different PPPs have to be included in the RA. Consequently, the inhibition of PPP1, PPP2A and PPP5 was determined with 18 structurally different MC and demonstrated MC congener dependent inhibition activity and a lower susceptibility of PPP5 to inhibition than PPP1 and PPP2A. The latter data were employed to train a machine learning algorithm that should allow prediction of PPP inhibition (toxicity) based on MCs 2D chemical structure. IC50 values were classified in toxicity classes and three machine learning models were used to predict the toxicity class, resulting in 80-90% correct predictions.

Simultaneous Detection of 14 Microcystin Congeners from Tissue Samples Using UPLC- ESI-MS/MS and Two Different Deuterated Synthetic Microcystins as Internal Standards.

Cyanobacterial microcystins (MCs), potent serine/threonine-phosphatase inhibitors, pose an increasing threat to humans. Current detection methods are optimised for water matrices with only a few MC congeners simultaneously detected. However, as MC congeners are known to differ in their toxicity, methods are needed that simultaneously quantify the congeners present, thus allowing for summary hazard and risk assessment. Moreover, detection of MCs should be expanded to complex matrices, e.g., blood and tissue samples, to verify in situ MC concentrations, thus providing for improved exposure assessment and hazard interpretation. To achieve this, we applied two synthetic deuterated MC standards and optimised the tissue extraction protocol for the simultaneous detection of 14 MC congeners in a single ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) run. This procedure was validated using plasma and liver homogenates of mice (male and female) spiked with deuterated MC standards. For proof of concept, tissue and plasma samples from mice i.p. injected with MC-LR and MC-LF were analysed. While MC-LF was detected in all tissue samples of both sexes, detection of MC-LR was restricted to liver samples of male mice, suggesting different toxicokinetics in males, e.g., transport, conjugation or protein binding. Thus, deconjugation/-proteinisation steps should be employed to improve detection of bound MC.

Adsorption of Ten Microcystin Congeners to Common Laboratory-Ware Is Solvent and Surface Dependent.

Cyanobacteria can produce heptapetides called microcystins (MC) which are harmful to humans due to their ability to inhibit cellular protein phosphatases. Quantitation of these toxins can be hampered by their adsorption to common laboratory-ware during sample processing and analysis. Because of their structural diversity (>100 congeners) and different physico-chemical properties, they vary in their adsorption to surfaces. In this study, the adsorption of ten different MC congeners (encompassing non-arginated to doubly-arginated congeners) to common laboratory-ware was assessed using different solvent combinations. Sample handling steps were mimicked with glass and polypropylene pipettes and vials with increasing methanol concentrations at two pH levels, before analysis by liquid chromatography-tandem mass spectrometry. We demonstrated that MC adsorb to polypropylene surfaces irrespective of pH. After eight successive pipet actions using polypropylene tips ca. 20% of the MC were lost to the surface material, which increased to 25%-40% when solutions were acidified. The observed loss was alleviated by changing the methanol (MeOH) concentration in the final solvent. The required MeOH concentration varied depending on which congener was present. Microcystins only adsorbed to glass pipettes (loss up to 30% after eight pipet actions) when in acidified aqueous solutions. The latter appeared largely dependent on the presence of ionizable groups, such as arginine residues.

Total Synthesis of Microcystin-LF and Derivatives Thereof.

Microcystins (MCs) are highly toxic natural products which are produced by cyanobacteria. They can be released to the water during harmful algal blooms and are a serious threat to animals and humans. Described is the total synthesis of the cyanotoxin microcystin-LF (MC-LF, 1a) and two derivatives thereof. Deuterated derivative 1b is of interest as an internal standard during MC quantification in biological samples by mass spectrometry and alkyne-labeled 1c can be employed for toxin derivatization by click chemistry with an azide-containing reporter molecule or as an activity-based probe to identify interaction partners. Application of tert-butyl ester protecting groups for erythro-ß-d-methylaspartic acid and γ-d-glutamic acid were key for an isomerization-free synthesis. The analytical data of synthetic MC-LF were identical to those of an authentic sample of the natural product. All derivatives 1a-c were determined to be potent inhibitors of protein phosphatase-1 with similar activity.

Comparison of two ELISA-based methods for the detection of microcystins in blood serum.

Microcystins (MCs) are cyanobacterial toxins which place the public at risk via exposure to MC contaminated water, food or algal food supplements. Subsequent to the fatal intravenous exposure of dialysis patients in Caruaru, Brazil, several techniques (LC-MS, GC-MS and ELISA) were adapted to detect MCs in human serum. As patients chronically exposed to low concentrations of MCs also present with very low MC serum levels, only LC-MS methodology would appear to allow detection of these MC levels. However, LC-MS detection depends on the availability of respective MC congener standards and the levels of non-covalently bound MC in the sample. In contrast, immunological techniques, e.g. MC-ELISA potentially could detect even covalently bound MC, provided the MC-antibody was raised against an epitope found in nearly all of the MC congeners. As the Adda-side-chain moiety is present in nearly all of the MC congeners known to date, the anti-Adda antibodies, when applied in Adda-ELISAs, could represent a relatively simple and robust technique for the qualitative and quantitative determination of MC in human serum. The aim of the current study was to determine whether commercially available Adda-ELISAs and their respective sample preparation methods would allow MC quantification in human serum. The Adda-ELISA (polyclonal antibody) and the Adda-ELISA (monoclonal antibody) kit for serum (Serum-ELISA) were used for determination of the concentration-dependent recovery of MCs in MC-spiked serum. Human serum samples were spiked with varying concentrations of MCs (MC-LR, -YR, -RR, -LA, -LW, -LF and defined MC mixtures) and extracted using two different methods. MC-spiked bovine serum and standard cell culture medium containing 10% FBS served to investigate potential matrix effects. Inter-laboratory comparison was performed allowing identification of potential sources of error. The results suggest that both ELISAs are suitable tools for the analysis of MCs in human blood serum although both also displayed some weaknesses notably the time needed for sample preparation or the overestimation of some specific MC congener concentrations. Based on the ELISA detection ranges, sample concentration and/or MC spiking may be required for detection of low levels of MCs in human blood.

Pitfalls in microcystin extraction and recovery from human blood serum.

BACKGROUND: Microcystins (MCs) contaminate water bodies due to cyanobacterial blooms all over the world, leading to frequent exposure of humans to MCs through consumption of meat, fish, seafood, blue-green algal products and water, accidental ingestion of contaminated water and scum during recreational activities and inhalation of cyanobacterial aerosols. For monitoring of human exposure, sensitive screening methods are needed. However, during the analytical process of various matrices, such as human serum, some problems appear to regularly occur during sample preparation and storage, leading to MC loss and thus to underestimation of the true MC concentration. The aim of the current study was therefore to assess the pitfalls of the MC-extraction method from human serum with more detail. METHODS: Six MC congeners (MC-LR, -YR, -RR, -LA, -LW, -LF) and defined equimolar MC mixtures thereof were spiked into human serum, and quantified using the commercially available Adda-ELISA subsequent to standard extraction (methanol extraction with subsequent SPE). To detect the potential influence of sample storage and preparation/storage materials, different types of material such as glass, standard polypropylene and surface-treated polypropylene were compared. RESULTS: Loss of MC during preparation and storage is largely dependent on (1) the handling of the stored material, (2) the 'surface' of the storage material and (3) the hydrophobicity of the MCs. CONCLUSIONS: The pitfalls described for MC analysis with the ELISA are primarily associated with sample preparation and clean-up and thus also apply to other analytical techniques for MC detection beyond the ELISA used. It can be concluded that ELISA-based methods are suitable tools for the detection of MCs in human sera and other samples.