Assessment of estrogenic potential from exudates of microcystin-producing and non-microcystin-producing Microcystis by metabolomics, machine learning and E-screen assay.

Cyanobacterial blooms, often dominated by Microcystis aeruginosa, are capable of producing estrogenic effects. It is important to identify specific estrogenic compounds produced by cyanobacteria, though this can prove challenging owing to the complexity of exudate mixtures. In this study, we used untargeted metabolomics to compare components of exudates from microcystin-producing and non-microcystin-producing M. aeruginosa strains that differed with respect to their ability to produce microcystins, and across two growth phases. We identified 416 chemicals and found that the two strains produced similar components, mainly organoheterocyclic compounds (20.2%), organic acids and derivatives (17.3%), phenylpropanoids and polyketides (12.7%), benzenoids (12.0%), lipids and lipid-like molecules (11.5%), and organic oxygen compounds (10.1%). We then predicted estrogenic compounds from this group using random forest machine learning. Six compounds (daidzin, biochanin A, phenylethylamine, rhein, o-Cresol, and arbutin) belonging to phenylpropanoids and polyketides (3), benzenoids (2), and organic oxygen compound (1) were tested and exhibited estrogenic potency based upon the E-screen assay. This study confirmed that both Microcystis strains produce exudates that contain compounds with estrogenic properties, a growing concern in cyanobacteria management.

Harmful and beneficial properties of cyanotoxins: Two sides of the same coin.

Cyanotoxins are by definition "harmful agents" produced by cyanobacteria. Their toxicity has been extensively studied and reviewed over the years. Cyanotoxins have been commonly classified, based on their poisonous effects on mammals, into three main classes, neurotoxins, hepatotoxins and dermatotoxins, and, considering their chemical features, mainly identified as peptides, alkaloids and lipopolysaccharides. Here we propose a broader subdivision of cyanotoxins into eight distinct classes, taking into account their molecular structures, biosynthesis and modes of action: alkaloids, non-ribosomal peptides, polyketides, non-protein amino acids, indole alkaloids, organophosphates, lipopeptides and lipoglycans. For each class, the structures and primary mechanisms of toxicity of the main representative cyanotoxins are reported. Despite their powerful biological activities, only recently scientists have considered the biotechnological potential of cyanotoxins, and their applications both in medical and in industrial settings, even if only a few of these have reached the biotech market. In this perspective, we discuss the potential uses of cyanotoxins as anticancer, antimicrobial, and biocidal agents, as common applications for cytotoxic compounds. Furthermore, taking into account their mechanisms of action, we describe peculiar potential bioactivities for several cyanotoxin classes, such as local anaesthetics, antithrombotics, neuroplasticity promoters, immunomodulating and antifouling agents. In this review, we aim to stimulate research on the potential beneficial roles of cyanotoxins, which require interdisciplinary cooperation to facilitate the discovery of innovative biotechnologies.

How UV radiation and pH alternation impact graphene oxide mediated environmental toxicant adsorption and resulting safety characteristics - A toxicology study beyond a classic carrier effect.

Once released into water, the widely used graphene oxide (GO) is likely to adsorb classical environmental pollutants, exemplified by Microcystin-LR (MCLR) that is a representative double-bond rich liver-toxic endotoxin. While GO-mediated carrier effect is fairly predictable, the involvement of environmental factors like UV and pH may add additional level of sophistication as these factors may impact the adsorption capacity of GO to MCLR. Here, we firstly investigated the changes of GO structure under different UV-radiation durations and pH conditions with a view to establish the correlation in terms of MCLR adsorption onto GO. We demonstrated that GO reduction especially oxygen-containing groups reduction induced by UV- radiation caused the compromised adsorption MCLR capacity on GO. Besides, the higher pH decreased the non-biological MCLR adsorption to GO by reducing GO defect sites and increasing electrostatic repulsion. These abiotic discoveries were further investigated to compare the safety features of GO-MCLR complex. Under dark condition (pH = 7), we revealed the cytotoxicity of GO-MCLR to normal liver cells, which involved the ROS generation and cell ferroptosis caused by Fe2+ accumulation. Introduction of UV and pH alternation in environment impacted GO-mediated environmental toxicant adsorption and resulting safety characteristics, which reminded us environmental factors should not be ignored in the GO-mediated carrier effect.

A novel ternary heterogeneous TiO2/BiVO4/NaY-Zeolite nanocomposite for photocatalytic degradation of microcystin-leucine arginine (MC-LR) under visible light.

Microcystin-leucine arginine (MC-LR) is a carcinogenic toxin, produced by cyanobacteria. The release of this toxin into drinking water sources can threaten public health and environmental safety. Therefore, effective MC-LR removal from water resources is necessary. In the present study, the hydrothermal method was used to synthesize a novel ternary BiVO4/TiO2/NaY-Zeolite (B/T/N-Z) nanocomposite for MC-LR degradation under visible light. FESEM, FTIR, XRD, and DRS were performed for characterizing the nanocomposite structure. Also, the Response Surface Methodology (RSM) was applied to determine the impact of catalyst dosage, pH, and contact time on the MC-LR removal. High-performance liquid chromatography was performed to measure the MC-LR concentration. Based on the results, independent parameters, including contact time, catalyst dosage, and pH, significantly affected the MC-LR removal (P < 0.05). In other words, increasing the contact time, catalyst dosage, and acidic pH had positive effects on MC-LR removal. Among these variables, the catalyst dosage, with the mean square and F-value of 1041.37 and 162.84, respectively, had the greatest effect on the MC-LR removal efficiency. Apart from the interaction between the catalyst dosage and contact time, the interaction effects of other parameters were not significant. Also, the maximum MC-LR removal efficiency was 99.88% under optimal conditions (contact time = 120 min, catalyst dosage = 1 g/L, and pH = 5). According to the results, the B/T/N-Z nanocomposite, as a novel and effective photocatalyst could be used to degrade MC-LR from polluted water.

Genotoxic Effects of Cylindrospermopsin, Microcystin-LR and Their Binary Mixture in Human Hepatocellular Carcinoma (HepG2) Cell Line.

Simultaneous occurrence of cylindrospermopsin (CYN) and microcystin-LR (MCLR) has been reported in the aquatic environment and thus human exposure to such mixtures is possible. As data on the combined effects of CYN/MCLR are scarce, we aimed to investigate the adverse effects related to genotoxic activities induced by CYN (0.125, 0.25 and 0.5 µg/mL) and MCLR (1 µg/mL) as single compounds and their combinations in HepG2 cells after 24 and 72 h exposure. CYN and CYN/MCLR induced DNA double-strand breaks after 72 h exposure, while cell cycle analysis revealed that CYN and CYN/MCLR arrested HepG2 cells in G0/G1 phase. Moreover, CYN and the combination with MCLR upregulated CYP1A1 and target genes involved in DNA-damage response (CDKN1A, GADD45A). Altogether, the results showed that after 72 h exposure genotoxic activity of CYN/MCLR mixture was comparable to the one of pure CYN. On the contrary, MCLR (1 µg/mL) had no effect on the viability of cells and had no influence on cell division. It did not induce DNA damage and did not deregulate studied genes after prolonged exposure. The outcomes of the study confirm the importance of investigating the combined effects of several toxins as the effects can differ from those induced by single compounds.

Synthesis of metal-organic framework-5@chitosan material for the analysis of microcystins and nodularin based on ultra-performance liquid chromatography-tandem mass spectrometry.

Microcystins (MCs) and nodularin (NOD) are tumor promoters produced by cyanobacteria and present in surface water. In this work, a novel mesoporous metal-organic framework-5@chitosan (MOF-5@CS) material was synthesized and applied for the enrichment of MCs and NOD in water and fish samples. The mesoporous MOF-5@CS material was firstly synthesized via a one-step hydrothermal method, and the chitosan was combined with MOF-5 via chemical bonding assembly. As a new adsorbent, the as-synthesized material was found having a large specific surface area and good thermal stability. Under the optimized conditions, MCs and NOD were enriched by the MOF-5@CS material and detected by ultra-performance liquid chromatography-tandem mass spectrometry. The limit of detection of the new method for MCs and NOD were in the range of 0.0018-0.077 ng/mL. The value of relative standard deviation for repeatability were 2.69-6.30%, and the recovery of the analytes ranged from 84.36% to 118.51%. Compared with other reported method for MCs and NOD detection in complex matrices, better adsorption performance for MCs and NOD were obtained by our new method, and the sensitivity of MCs-RR and NOD were improved nearly 20 times and 30 times, respectively.

Precursor-Directed Biosynthesis and Fluorescence Labeling of Clickable Microcystins.

Microcystins, cyclic nonribosomal heptapeptides, are the most well-known cyanobacterial toxins. They are exceptionally well studied, but open questions remain concerning their physiological role for the producing microorganism or their suitability as lead compounds for anticancer drug development. One means to study specialized metabolites in more detail is the introduction of functional groups that make a compound amenable for bioorthogonal, so-called click reactions. Although it was reported that microcystins cannot be derivatized by precursor-directed biosynthesis, we successfully used this approach to prepare clickable microcystins. Supplementing different azide- or terminal alkyne containing amino acid analogues into the cultivation medium of microcystin-producing cyanobacteria strains, we found that these strains differ strongly in their substrate acceptance. Exploiting this flexibility, we generated more than 40 different clickable microcystins. We conjugated one of these derivatives with a fluorogenic dye and showed that neither incorporation of the unnatural amino acid analogue nor attachment of the fluorescent label significantly affects the cytotoxicity against cell lines expressing the human organic anion transporting polypeptides 1B1 or 1B3. Using time-lapse microscopy, we observed that the fluorescent microcystin is rapidly taken up into eukaryotic cells expressing these transporters.

A Mini Review on Microcystins and Bacterial Degradation.

Microcystins (MCs) classified as hepatotoxic and carcinogenic are the most commonly reported cyanobacterial toxins found in the environment. Microcystis sp. possessing a series of MC synthesis genes (mcyA-mcyJ) are well documented for their excessive abundance, numerous bloom occurrences and MC producing capacity. About 246 variants of MC which exert severe animal and human health hazards through the inhibition of protein phosphatases (PP1 and PP2A) have been characterized. To minimize and prevent MC health consequences, the World Health Organization proposed 1 µg/L MC guidelines for safe drinking water quality. Further the utilization of bacteria that represent a promising biological treatment approach to degrade and remove MC from water bodies without harming the environment has gained global attention. Thus the present review described toxic effects and bacterial degradation of MCs.

Efficiency and mechanisms of simultaneous removal of Microcystis aeruginosa and microcystins by electrochemical technology using activated carbon fiber/nickel foam as cathode material.

The significant removal efficiency of microcystis aeruginosa was presented using Pt/Ti anode and activated carbon fiber/nickel foam (ACF/Ni) cathode by addition of Fe2+ slightly in a wide range of initial pH (3-9). Results showed that about 93% of the Microcystis aeruginosa cells were removed within 15 min for Pt/Ti-ACF/Ni-Fe2+ system. Dosage of Fe2+, current density, and initial pH had remarkable effects on the removal efficiency of microcystis aeruginosa. The mechanism of algae removal in the Pt/Ti-ACF/Ni-Fe2+ electrochemical system was revealed by the comparison between Pt/Ti-ACF/Ni-Fe2+ process and classical Fenton process, the analysis on Microcystis aeruginosa and ACF/Ni by SEM, the specific surface area and pore size analysis of ACF, and the determination of UV254, OD620 and microcystin-LR (MC-LR). Results showed that the main mechanism of this system was the electro-Fenton process, which was accompanied by electro-adsorption, electro-floatation, and electro-coagulation process. And the cooperation mechanism on the electrochemical removal system was further speculated. With the breakdown of algal cells during the electrolysis, the MC-LR and other substances released from the cells were effectively degraded. Besides, the new cathode exhibited favorable and stable reusability. This study built up a high-efficiency algae removal system, which broke through the limits of narrow working pH range and large consumption of exogenous chemicals in electro-Fenton process.

In vitro detoxication of microcystins in human samples: variability among variants with different hydrophilicity and structure.

Cyanotoxins, among which >200 variants of Microcystins (MC), constitute an emerging issue in food safety. Microcystins (MC) toxicity is congener-specific; however, the in vitro inhibition of PP1/PP2A (the key molecular event of MC toxicity) by single MC variants is comparable and MC toxicokinetics seems to be the critical point. Here, the variability in GSH conjugation catalysed by human recombinant enzymes and human hepatic cytosol has been compared between hydrophilic (MC-LR and MC-RR) and hydrophobic (MC-LW, MC-YR and MC-LF) variants, according to measured logPow. In vitro detoxication reaction (spontaneous plus enzymatic) is favored by the variant hydrophilicity, with MC-LF very poorly detoxified. With MC-YR and -LW the spontaneous reaction always gave the major contribution, whereas with MC-LR and -RR the enzymatic reaction became by far predominant when GSH was depleted. Consequently, the well-known GST polymorphisms seems not to be the major driver for potential human variability in susceptibility towards the MC-toxicity, except for MC-RR and -LR when GSH is depleted. Looking at these results and literature data, MC-RR (the least cytotoxic and acutely toxic in rodents) is the more hydrophilic, has the lowest OATP-mediated hepatic uptake and the highest detoxication efficiency. The opposite is true for the most lipophilic MC-LF: once entered in the cells with the highest uptake, it is very poorly detoxified, and resulted as the most toxic in various cell types. MC-dependent TK should be considered in order to estimate the variability in toxicity and to support the use of quantitative in vitro-in vivo extrapolation models of single toxins and their mixtures co-occurring in the environment.

Effect of the immobilized microcystin-LR-degrading enzyme MlrA on nodularin degradation and its immunotoxicity study.

In freshwater ecosystems with frequent cyanobacterial blooms, the cyanobacteria toxin pollution is becoming increasingly serious. Nodularin (NOD), which has strong biological toxicity, has emerged as a new pollutant and affects the normal growth, development and reproduction of aquatic organisms. However, little information is available regarding this toxin. In this study, a graphene oxide material modified by L-cysteine was synthesized and used to immobilize microcystin-LR (MC-LR)-degrading enzyme (MlrA) to form an immobilized enzyme nanocomposite, CysGO-MlrA. Free-MlrA was used as a control. The efficiency of NOD removal by CysGO-MlrA was investigated. Additionally, the effects of CysGO-MlrA and the NOD degradation product on zebrafish lymphocytes were detected to determine the biological toxicity of these two substances. The results showed the following: (1) There was no significant difference in the degradation efficiency of NOD between CysGO-MlrA and free-MlrA; the degradation rate of both was greater than 80% at 1 h (2) The degradation efficiency of the enzyme could retain greater than 81% of the initial degradation efficiency after the CysGO-MlrA had been reused 7 times. (3) CysGO-MlrA retained greater than 50% of its activity on the 8th day when preserved at 0 °C, while free-MlrA lost 50% of its activity on the 4th day. (4) CysGO-MlrA and the degradation product of NOD showed no obvious cytotoxicity to zebrafish lymphocytes. Therefore, CysGO-MlrA might be used as an efficient and ecologically safe degradation material for NOD.

A Data-Independent Methodology for the Structural Characterization of Microcystins and Anabaenopeptins Leading to the Identification of Four New Congeners.

Toxin-producing cyanobacteria are responsible for the presence of hundreds of bioactive compounds in aquatic environments undergoing increasing eutrophication. The identification of cyanotoxins is still emerging, due to the great diversity of potential congeners, yet high-resolution mass spectrometry (HRMS) has the potential to deepen this knowledge in aquatic environments. In this study, high-throughput and sensitive on-line solid-phase extraction ultra-high performance liquid chromatography (SPE-UHPLC) coupled to HRMS was applied to a data-independent acquisition (DIA) workflow for the suspect screening of cyanopeptides, including microcystin and anabaenopeptin toxin classes. The unambiguous characterization of 11 uncommon cyanopeptides was possible using a characterization workflow through extensive analysis of fragmentation patterns. This method also allowed the characterization of four unknown cyanotoxins ([Leu1, Ser7] MC-HtyR, [Asp3]MC-RHar, AP731, and AP803). The quantification of 17 common cyanotoxins along with the semi-quantification of the characterized uncommon cyanopeptides resulted with the identification of 23 different cyanotoxins in 12 lakes in Canada, United Kingdom and France. The concentrations of the compounds varied between 39 and 41,000 ng L-1. To our knowledge, this is the first DIA method applied for the suspect screening of two families of cyanopeptides simultaneously. Moreover, this study shows the great diversity of cyanotoxins in lake water cyanobacterial blooms, a growing concern in aquatic systems.

Novel Microcystins from Planktothrix prolifica NIVA-CYA 544 Identified by LC-MS/MS, Functional Group Derivatization and 15N-labeling.

Microcystins are cyclic heptapeptides from cyanobacteria that are potent inhibitors of protein phosphatases and are toxic to animals and humans. At present, more than 250 microcystin variants are known, with variants reported for all seven peptide moieties. While d-glutamic acid (d-Glu) is highly-conserved at position-6 of microcystins, there has been only one report of a cyanobacterium (Anabaena) producing microcystins containing l-Glu at the variable 2- and 4-positions. Liquid chromatography-mass spectrometry analyses of extracts from Planktothrix prolifica NIVA-CYA 544 led to the tentative identification of two new Glu-containing microcystins, [d-Asp3]MC-ER (12) and [d-Asp3]MC-EE (13). Structure determination was aided by thiol derivatization of the Mdha7-moiety and esterification of the carboxylic acid groups, while 15N-labeling of the culture and isotopic profile analysis assisted the determination of the number of nitrogen atoms present and the elemental composition of molecular and product-ions. The major microcystin analog in the extracts was [d-Asp3]MC-RR (1). A microcystin with an unprecedented high-molecular-mass (2116 Da) was also detected and tentatively identified as a sulfide-linked conjugate of [d-Asp3]MC-RR (15) by LC-HRMS/MS and sulfide oxidation, together with its sulfoxide (16) produced via autoxidation. Low levels of [d-Asp3]MC-RW (14), [d-Asp3]MC-LR (4), [d-Asp3,Mser7]MC-RR (11), [d-Asp3]MC-RY (17), [d-Asp3]MC-RF (18), [d-Asp3]MC-RR-glutathione conjugate (19), and [d-Asp3]MC-RCit (20), the first reported microcystin containing citrulline, were also identified in the extract, and an oxidized derivative of [d-Asp3]MC-RR and the cysteine conjugate of 1 were partially characterized.

Comprehensive multi-technique approach reveals the high diversity of microcystins in field collections and an associated isolate of Microcystis aeruginosa from a Turkish lake.

Microcystins (MCs) are hepatotoxic and potentially carcinogenic cyanotoxins. They exhibit high structural variability, with nearly 250 variants described to date. This variability can result in incomplete detection of MC variants during lake surveys due to the frequent use of targeted analytical methods and a lack of standards available for identification and quantitation. In this study, Lake Uluabat in Turkey was sampled during the summer of 2015. Phylogenetic analysis of the environmental mcyA sequences suggested Microcystis spp. were the major MC contributors. A combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography with UV detection and mass spectrometry (LC-UV-MS), and a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) method, together with thiol and periodate reactivity, revealed more than 36 MC variants in the lake samples and a strain of M. aeruginosa (AQUAMEB-24) isolated from Lake Uluabat. Only MCs containing arginine at position-4 were detected in the culture, while MC-LA, -LY, -LW and -LF were also detected in the lake samples, suggesting the presence of other MC producers in the lake. The previously unreported MCs MC-(H2)YR (dihydrotyrosine at position-2) (17), [epoxyAdda5]MC-LR, [DMAdda5]MC-RR (1) and [Mser7]MC-RR (8) were detected in the culture and/or field samples. This study is a good example of how commonly used targeted LC-MS methods can underestimate the diversity of MCs in freshwater lakes and cyanobacteria cultures and how untargeted LC-MS methods can be used to comprehensively assess MC diversity present in a new system.

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.

Stable-isotope dilution LC-MS/MS method for quantitative determination of microcystin conjugates with cysteine and glutathione in biotic matrices.

Microcystins are cyclic peptide toxins with hepatotoxic and tumor-promoting properties, which are produced in significant quantities (up to tens of µg/L) in freshwater cyanobacterial water blooms. Several studies reported microcystin accumulation in fish with possible food transfer to humans. These compounds are further metabolized to cysteine and glutathione conjugates which can be present in tissues in significant concentrations. In this study, we focused on the development and evaluation of robust and highly sensitive SPE-LC-MS/MS method for the analysis of microcystin conjugates in fish tissue samples. For the first time, we demonstrate the use of isotopically labeled internal standards which are essential for accurate and precise determination of analytes in complex biotic matrices. LLOQs of respective microcystin conjugates (signal-to-noise ratio; S/N > 10, peak-to-peak method) ranged from 3.3 to 5.0 ng/g of tissue fresh weight (FW). The calibration was linear within a range of concentrations from 1 to 70 ng/mL for all analyzed conjugates. The precision and repeatability of the method were very good with recoveries in the range of 88.5-107.6% and relative standard deviations between 8.8 and 13.2% for all analytes. In the follow-up study, fully validated method was used for the determination of microcystin conjugate levels in common carp exposed to microcystin-containing cyanobacterial biomass under controlled conditions. Significant amounts of microcystin conjugates (up to 55 ng/g) were found in the tissues of fish after 7 weeks of exposure. Our method was shown to be robust, sensitive, selective, and suitable for the determination of trace levels of microcystin conjugates in fish tissues.

Genotoxicity and cytotoxicity of three microcystin-LR containing cyanobacterial samples from Antioquia, Colombia.

The presence of cyanobacterial blooms and cyanotoxins in water presents a global problem due to the deterioration of ecosystems and the possibility of poisoning in human and animals. Microcystin LR is the most widely distributed cyanotoxin and liver cells are its main target. In the present study, HepG2 cells were used to determine DNA damage of three crude extracts of cyanobacterial blooms containing MC-LR, through comet assay. The results show that all extracts at a concentration of 500 µg mL-1 caused low damage in hepatocytes exposed for 24 h, but produced total mortality even at low concentrations at 48 h. Moreover, balloons corresponding to cell apoptosis were found. Through HPLC/MS, MC-LR was detected in all samples of cyanobacterial blooms at concentrations of (5,65 µg ml-1) in sample 1, (1,24 µg ml-1) in sample 2 and (57,29 µg ml-1) in sample 3. In addition, in all samples high molecular weights peaks were detected, that may correspond to other microcystins. Besides, the cytotoxic effect of a cyanobacterial bloom and some of its chromatographic fractions from the crude extracts were evaluated in U-937, J774, Hela and Vero cell lines, using the enzymatic micromethod (MTT). The highest toxicity was detected in U-937 cells (LC50 = 29.7 µg mL-1) and Vero cells (LC50 = 39.7 µg mL-1). Based on these results, it is important to remark that genotoxic and cytotoxicity assays are valuable methods to predict potential biological risks in waters contaminated with blooms of cyanobacteria, since chemical analysis can only describe the presence of cyanotoxins, but not their biological effects.

Chronic exposure to microcystin-leucine-arginine promoted proliferation of prostate epithelial cells resulting in benign prostatic hyperplasia.

Microcystin-leucine-arginine (MC-LR), as a most common and deleterious variant among all structural analogues of Microcystins (MCs), can cause male reproductive dysfunction. However, its toxic effects on prostate in adult mice have not been invested in detail. In this study, we observed that MC-LR could enter prostate tissues and induce focal hyperplasia and prostate inflammation. Moreover, increased levels of prostate specific antigen (PSA) and prostate acid phosphatase (PAP) in serum of mice following chronic exposure to MC-LR were detected. We also examined increased expression of forkhead box protein M1 (FOXM1) and PSA in human prostate epithelial cells (RWPE-1) treated with MC-LR at low levels, and FOXM1 could regulate PSA expression. Furthermore, MC-LR also induced expression of CyclinD1 via FOXM1/Wnt/ß-catenin signaling pathways in RWPE-1 cells, promoting proliferation of prostate epithelial cells, resulting in prostatic hyperplasia in vivo. As a foreign substance, MC-LR also induced immune reaction in RWPE-1 cells mediated by NF-κB pathway, promoting production of pro-inflammatory cytokines and chemokines. Collectively, these findings demonstrated that MC-LR may induce prostatic hyperplasia and prostatitis in mice following chronic low-dose exposure to MC-LR. This work may provide new perspectives in developing new diagnosis and treatment strategies for MC-LR-induced prostatic toxicity.

Microcystins Containing Doubly Homologated Tyrosine Residues from a Microcystis aeruginosa Bloom: Structures and Cytotoxicity.

Four new microcystin congeners are described including the first three examples of microcystins containing the rare doubly homologated tyrosine residue 2-amino-5-(4-hydroxyphenyl)pentanoic acid (Ahppa) (1-4). Large-scale harvesting and biomass processing allowed the isolation of substantial quantities of these compounds, thus enabling complete structure determination by NMR as well as cytotoxicity evaluation against selected cancer cell lines. The new Ahppa-toxins all incorporate Ahppa residues at the 2-position, and one of these also has a second Ahppa at position 4. The two most lipophilic Ahppa-containing microcystins showed 10-fold greater cytotoxic potency against human tumor cell lines (A549 and HCT-116) compared to microcystin-LR (5). The presence of an Ahppa residue in microcystin congeners is difficult to ascertain by MS methods alone, due to the lack of characteristic fragment ions derived from the doubly homologated side chain. Owing to their unexpected cytotoxic potency, the potential impact of the compounds on human health should be further evaluated.

Immunomodulatory effects of selected cyanobacterial peptides in vitro.

Cyanobacteria produce many biologically active metabolites synthesized via nonribosomal synthetic pathways such as cyclic microcystins (MCs) and linear aeruginosins (Aers). The present study aimed to investigate the effects of different MC variants and the newly isolated aerugenosin Aer-865 on macrophages, which represent one of the key effector cells within the innate immune responses. Specifically, our study included RAW 264.7 macrophage activation associated with production of cytotoxic and cytostatic nitric oxide (NO) as well as pro-inflammatory mediators like tumor necrosis factor α (TNFα) and interleukin 6 (IL-6). From the compounds investigated, commonly occurring MC variants (-RR, -YR) and Aer-865 had no significant effects within the non-cytotoxic concentrations tested, i.e. 0.001-1 µM for MCs and 0.1-50 µM for Aer-865. In contrast to known immunoactive MC-LR, the negligible immunomodulatory potential of tested MC congeners could be related to their differences in structure. The knowledge of MC structure-specific activities contributes to the understanding of complex toxicity of different MC variants and most importantly their mixtures. This study is one of the first study that evaluate the effect of larger set of cyanobacterial peptides on macrophages and compare their immunomodulatory potential.