cyanoginosin-lr and nodularin

Microcystin-LR and nodularin, along with okadaic acid, are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). The mechanisms of action of microcystin-LR and nodularin in the liver and that of okadaic acid, a potent tumor promoter on mouse skin, have attracted the attention of the scientists. This paper reviews several topics: new inhibitors of PP1 and PP2A with new chemical structures, structure-function relationships for both receptor binding and inhibition of protein phosphatases, the crystal structure of PP1 or PP2A-toxin complex, induction of gene expression and apoptosis. These subjects were studied by using in vitro and in vivo experimental systems. Two-stage carcinogenesis experiments with microcystin-LR and nodularin for the first time demonstrated that microcystin-LR is a new tumor promoter in rat liver initiated with diethylnitrosamine (DEN), and that nodularin is a potent tumor promoter associated with weak initiating activity in rat liver initiated with DEN. A working group of WHO (IARC) concluded that microcystin-LR is "possibly carcinogenic to humans" and that nodularin is "not classifiable as to carcinogenicity". Our studies revealed that chemical tumor promoters are inducers of TNF-α in the cells of target tissues and that TNF-α is an endogenous tumor promoter. This advance in carcinogenesis made it possible to look for the link between chemical tumor promoters and endogenous tumor promoters, such as TNF-α and IL-1. The carcinogenic features of TNF-α are described in this review, and the TNF-α inducing protein (Tipα) of Helicobacter pylori genome is presented as an example of a tumor promoter of human stomach cancer development.

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Humans; Marine Toxins; Microcystins; Neoplasms; Okadaic Acid; Peptides, Cyclic; Protein Tyrosine Phosphatases; Structure-Activity Relationship; Tumor Necrosis Factor-alpha

Iningainema is a recently described genus of heterocytous, false-branching cyanobacteria originally described from Australia. In this work, we present Iningainema tapete sp. nov., isolated from subaerial and terrestrial environments in central Florida (USA). In comparison to the sister species, our novel cyanobacterium produces nodularin-R (NOD-R) and a methylated isoform [MeAdda

Topics: Australia; Cyanobacteria; Florida; Marine Toxins; Microcystins; Peptides, Cyclic; RNA, Ribosomal, 16S

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.

Topics: Animals; Cyanobacteria; Ecosystem; Enzymes, Immobilized; Marine Toxins; Microcystins; Peptides, Cyclic

Topics: Bacterial Toxins; Chromatography, High Pressure Liquid; Cyanobacteria Toxins; Drinking Water; Fresh Water; Limit of Detection; Marine Toxins; Mass Spectrometry; Microcystins; Mycotoxins; Peptides, Cyclic; Solid Phase Extraction; Spain; Tropanes; Water Pollutants, Chemical

An analytical procedure is proposed for determining three cyanotoxins (microcystin RR, microcystin LR, and nodularin) and two phycotoxins (domoic and okadaic acids) in seawater and algae-based food supplements. The toxins were first isolated by a salting out liquid extraction procedure. Since the concentration expected in the samples was very low, a dispersive liquid-liquid microextraction procedure was included for preconcentration. The ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate (80 mg) was used as green extractant solvent and acetonitrile as disperser solvent (0.5 mL) for a 10 mL sample volume at pH 1.5, following the principles of green analytical chemistry. Liquid chromatography with electrospray ionization and quadrupole time of flight-mass spectrometry (LC-Q-TOF-MS) was used. The selectivity of the detection system, based on accurate mass measurements, allowed the toxins to be unequivocally identified. Mass spectra for quadrupole time of flight-mass spectrometry (Q-TOF-MS) and Q-TOF-MS/MS were recorded in the positive ion mode and quantification was based on the protonated molecule. Retention times ranged between 6.2 and 17.9 min using a mobile phase composed by a mixture of methanol and formic acid (0.1%). None of the target toxins were detected in any of the seawater samples analyzed, above their corresponding detection limits. However, microcystin LR was detected in the blue green alga sample.

Topics: Acetonitriles; Borates; Chromatography, High Pressure Liquid; Dietary Supplements; Food Contamination; Imidazoles; Ionic Liquids; Kainic Acid; Liquid Phase Microextraction; Marine Toxins; Microcystins; Okadaic Acid; Peptides, Cyclic; Seawater; Solvents; Spain; Spirulina; Stramenopiles; Tandem Mass Spectrometry

In the present work, the degradation of three cyanotoxins from the hepatotoxins group was investigated under laboratory-controlled experiments in water samples. Surface waters spiked with microcystin-LR (MC-LR), nodularin (NOD) and cylindrospermopsin (CYN) were subjected to hydrolysis, chlorination and photo-degradation, under both sunlight (SL) and ultraviolet (UV) radiation. A total of 12 transformation products (TPs) were detected and tentatively identified by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF MS). These comprised: 6 chlorination TPs (3 from CYN and 3 from MC-LR, 2 isomers); 4 UV TPs (all from CYN); and 2 sunlight TPs (one isomer from MC-LR and another from NOD). No TPs were observed under hydrolysis conditions. The chemical structures for all TPs were tentatively proposed based on the accurate-mass QTOF MS full-spectra. Analysis of real-world samples collected from the Peñol reservoir (Antioquia, Colombia) revealed the presence of MC-LR and CYN as well as a sunlight TP identified in the laboratory experiments. Data presented in this article will assist further research on TPs potentially formed in future tertiary degradation processes applied for the removal of organic micro-pollutants in water; as well as improving available knowledge on the toxic implications of cyanobacterial toxins TPs in surface waters.

Topics: Alkaloids; Bacterial Toxins; Chromatography, Liquid; Colombia; Cyanobacteria Toxins; Halogenation; Hydrolysis; Marine Toxins; Mass Spectrometry; Microcystins; Peptides, Cyclic; Sunlight; Ultraviolet Rays; Uracil; Water Pollutants, Chemical

Developing easy-to-use and miniaturized detectors is essential for in-field monitoring of environmentally hazardous substances, such as the cyanotoxins. We demonstrated a differential fluorescent sensor array made of aptamers and single-stranded DNA (ssDNA) dyes for multiplexed detection and discrimination of four common cyanotoxins with an ordinary smartphone within 5 min of reaction. The assay reagents were preloaded and dried in a microfluidic chip with a long shelf life over 60 days. Upon the addition of analyte solutions, competitive binding of cyanotoxin to the specific aptamer-dye conjugate occurred. A zone-specific and concentration-dependent reduction in the green fluorescence was observed as a result of the aptamer conformation change. The aptasensors are fully optimized by quantification of their dissociation constants, tuning the stoichiometric ratios of reaction mixtures, and implementation of an internal intensity correction step. The fluorescent sensor array allowed for accurate identification and measurement of four important cyanotoxins, including anatoxin-a (ATX), cylindrospermopsin (CYN), nodularin (NOD), and microcystin-LR (MC-LR), in parallel, with the limit of detection (LOD) down to a few nanomolar (<3 nM), which is close to the World Health Organization's guideline for the maximum concentration allowed in drinking water. The smartphone-based sensor platform also showed remarkable chemical specificity against potential interfering agents in water. The performance of the system was tested and validated with real lake water samples that were contaminated with trace levels of individual cyanotoxins as well as binary, ternary, and quaternary mixtures. Finally, a smartphone app interface has been developed for rapid on-site data processing and result display.

Topics: Alkaloids; Aptamers, Nucleotide; Bacterial Toxins; Biosensing Techniques; Cyanobacteria Toxins; DNA, Single-Stranded; Fluorescence; Fresh Water; Humans; Lab-On-A-Chip Devices; Lakes; Limit of Detection; Marine Toxins; Microarray Analysis; Microcystins; Peptides, Cyclic; Smartphone; Tropanes; Uracil; Water Pollutants, Chemical

Microcystins (MCs) are hepatotoxic cyanobacterial metabolites produced sporadically in aquatic environments under favorable environmental conditions. Affinity of these toxins to covalently bind with protein phosphatases poses a challenge in their detection. Lemieux oxidation to release 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB), a common moiety to all MCs congeners, has been used in detection of these compounds, however a lack of sensitivity has limited the usefulness of the method. In this study, modifications of the oxidation and solid phase extraction procedures, combined with a sensitive LC/MS/MS (liquid chromatography/mass spectrometry) detection, have resulted in 25 ng/g method detection limits in both liver and plasma samples. Samples harvested from six fingerling channel catfish (Ictalurus punctatus) dosed intraperitoneally with a sublethal MC-LR dose of 250 μg/kg were analyzed, and microcystin concentrations ranging from 370 to 670 ng/g in plasma and 566-1030 ng/g in liver were detected. Similarly, 250 μg/kg nodularin-dosed channel catfish fish were found to contain 835-1520 ng/g in plasma and 933-1140 ng/g in liver. Detection of the toxins in serum and liver combined with the presence of histopathological lesions consistent with these hepatocellular toxin in exposed fish and no positive findings in the control fish demonstrates the usefulness of this analytical procedure for the diagnosis of suspected algal toxicity cases.

Topics: Animals; Chromatography, Liquid; Ictaluridae; Kidney; Limit of Detection; Liver; Marine Toxins; Microcystins; Peptides, Cyclic; Solid Phase Extraction; Tandem Mass Spectrometry

Monitoring the quality of freshwater is an important issue for public health. In the context of the European project μAqua, 150 samples were collected from several waters in France, Germany, Ireland, Italy, and Turkey for 2 yr. These samples were analyzed using 2 multitoxin detection methods previously developed: a microsphere-based method coupled to flow-cytometry, and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. The presence of microcystins, nodularin, domoic acid, cylindrospermopsin, and several analogues of anatoxin-a (ATX-a) was monitored. No traces of cylindrospermopsin or domoic acid were found in any of the environmental samples. Microcystin-LR and microcystin-RR were detected in 2 samples from Turkey and Germany. In the case of ATX-a derivatives, 75% of samples contained mainly H

Topics: Alkaloids; Bacterial Toxins; Bridged Bicyclo Compounds, Heterocyclic; Chromatography, Liquid; Cyanobacteria; Cyanobacteria Toxins; Environmental Monitoring; Eutrophication; Flow Cytometry; France; Fresh Water; Germany; Italy; Limit of Detection; Marine Toxins; Microcystins; Molecular Structure; Peptides, Cyclic; Tandem Mass Spectrometry; Tropanes; Turkey; Uracil; Water Pollutants, Chemical

Microcystin (MC) and nodularin are structurally similar cyanobacterial toxins that inhibit protein phosphatases. Additional modes of action are poorly known, in particular for nodularin. In our associated work, we showed that active cellular uptake is mediated by the organic anion transporting polypeptide drOatp1d1 in zebrafish (Faltermann et al., 2016). Here, we assessed the transcriptional expression of three genes encoding three uptake transporters during embryonic development from 24h post fertilization (hpf) to 168 hpf. Transcripts of drOatp1d1 and drOatp2b1 are present at 24 hpf. The abundance increased after hatching and remained about constant up to 168 hpf. Transcripts of drOatp2b1 were most abundant, while drOapt1f transcripts showed very low relative abundance compared to drOatp1d1 and drOatp2b1. We further demonstrated the uptake of fluorescent labeled MC-LR in eleuthero-embryos and its accumulation in the glomerulus of the pronephros. An important molecular effect of MC-LR in human liver cells is the induction of endoplasmic reticulum (ER)-stress. Here, we investigated, whether MC-LR and nodularin similarly lead to induction of ER-stress in zebrafish by analyzing changes of mRNA levels of genes indicative of ER-stress. In zebrafish liver organ cultures short- and long-term exposures to 0.15 and 0.3 μmol L(-1) MC-LR, and 0.5 and 1 μM L(-1) nodularin led to significant transcriptional induction of several ER-stress marker genes, including the chaperone glucose regulated protein 78 (bip), the spliced form of x-box binding protein (xbp-1s), the CCAAT-enhancer-binding protein homologous protein (chop) and activating transcription factor 4 (atf4). Furthermore, strong transcriptional changes occurred for tumor necrosis factor alpha (tnfa) and dual specificity phosphatase 5 (dusp5), associated with mitogen activated protein kinase (MAPK) pathway. However, no alterations in transcript levels of pro-apoptotic genes Bcl-2 like protein 4 (bax) and p53 occurred. In contrast to adult liver, MC-LR and nodularin did not result in detectable changes of mRNA levels of selected target genes involved in ER-stress in zebrafish eleuthero-embryos, nor was the abundance of transcripts belonging to the MAPK and pro-apoptosis pathways altered. In conclusion, our data indicate that MC-LR and nodularin have similar transcriptional effects. They lead to changes in mRNA levels of genes that suggest induction of ER-stress, and furthermore, lead to increased level of tnfα m

Topics: Animals; Embryo, Nonmammalian; Endoplasmic Reticulum Stress; Gene Expression Regulation, Developmental; Humans; Liver; Marine Toxins; Microcystins; Peptides, Cyclic; Water Pollutants, Chemical; Zebrafish

Microcystin-LR (MC-LR) and nodularin are hepatotoxins produced by several cyanobacterial species. Their toxicity is based on active cellular uptake and subsequent inhibition of protein phosphatases PP1/2A, leading to hyperphosphorylation and cell death. To date, uptake of MC-LR and nodularin in fish is poorly understood. Here, we investigated the role of the organic anion transporting polypeptide Oatp1d1 in zebrafish (drOatp1d1, Slco1d1) in cellular uptake in zebrafish. We stably transfected CHO and HEK293 cell lines expressing drOatp1d1. In both transfectants, uptake of MC-LR and nodularin was demonstrated by competitive inhibition of uptake with fluorescent substrate lucifer yellow. Direct uptake of MC-LR was demonstrated by immunostaining, and indirectly by the high cytotoxicity in stable transfectants. By means of a synthesized fluorescent labeled MC-LR derivative, direct uptake was further confirmed in HEK293 cells expressing drOatp1d1. Additionally, uptake and toxicity was investigated in the permanent zebrafish liver cell line ZFL. These cells had only a low relative abundance of drOatp1d1, drOatp2b1 and drOatp1f transcripts, which correlated with the lack of MC-LR induced cytotoxicity and transcriptional changes of genes indicative of endoplasmic reticulum stress, a known effect of this toxin. Our study demonstrates that drOatp1d1 functions as an uptake transporter for both MC-LR and nodularin in zebrafish.

Topics: Animals; Cyanobacteria; HEK293 Cells; Humans; Liver; Marine Toxins; Microcystins; Organic Anion Transporters; Peptides, Cyclic; Transfection; Water Pollutants, Chemical; Zebrafish

Microcystins have been detected in raw and finished drinking water using a variety of techniques, including assays (immunoassay, phosphatase inhibition) and HPLC (UV, MS/(MS)). The principal challenge to microcystin analysis is accounting for the over 150 variants that have been described. A confirmatory individual variant HPLC analysis is prone to under-reporting total microcystins due to method specificity. One method that allows for total microcystin quantitation is the MMPB technique. In this study, water samples with native microcystins were oxidized to cleave the Adda moiety, common to all microcystin variants. LC-MS/MS analysis was conducted on the subsequent MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) molecule and calibrated using a certified reference standard (microcystin-LR) and 4-phenylbutyric acid. Total microcystin concentrations from MMPB were compared to Adda ELISA and individual variant analyses (LC-UV, LC-MS/(MS)). Variants of microcystin, including [DAsp(3)]MC-RR, [Dha(7)]MC-RR, MC-RR, MC-YR, MC-LR, [DAsp(3)]MC-LR, [Dha(7)]MC-LR, MC-WR, MC-LA, and MC-LY were detected and quantified in samples. The individual variant analyses did not account for total microcystins present in samples, as indicated by ELISA and MMPB data. Results demonstrated the MMPB technique is a simple and valuable approach to confirm ELISA data when analyzing microcystins, with method detection limits of 0.05 μg L(-1) for total microcystins.

Topics: Chromatography, High Pressure Liquid; Drinking Water; Marine Toxins; Microcystins; Peptides, Cyclic; Phenylbutyrates; Tandem Mass Spectrometry; Water Microbiology

In countries around the Baltic Sea grazing ruminants have access to and drink, surface water from lakes, rivers and in several coastal regions. The water quality of these naturally occurring reservoirs affects performance and health of livestock. In the Baltic Sea both microcystin (MC) and nodularin (NOD) occurs as cyclic peptides and have hepatotoxic effects. Although cattle obviously have died after consuming contaminated water very little information is available as to how susceptible ruminants are to the toxins produced by cyanobacteria. The critical question as to whether the rumen microflora might constitute a protective shield is unresolved. For this reason our aim is to investigate a possible degradation rate of these toxins in rumen.. The ability of rumen microorganisms to degrade certain important cyanotoxins (MC-LR, YR, RR and NOD) was studied in vitro by incubating with rumen fluid at three different concentrations (0.05, 0.5 and 5 μg/mL) for 3 h. The degradation efficiencies were determined by LC-MS (ESI) positive mode. Degradation was observed in the following order MC-RR 36%, NOD 35%, MC-RR 25% and MC-LR 8.9% at lower concentrations within 3 h. However, average degradation was observed at concentration of 0.5 μg/mL. No degradation was observed in higher concentrations for entire 3 h. The present results reveal that the degradation was both dose and time dependent.. In conclusion the present results suggest that the rumen microbial flora may protect ruminants from being intoxicated by Cyanotoxins.

Topics: Animals; Body Fluids; Cattle; Female; Marine Toxins; Microcystins; Peptides, Cyclic; Rumen

We describe within this paper the development of an affinity sensor for the detection of the cyanobacterial toxin microcystin-LR. The first stage of the work included acquiring and testing of the antibodies to this target. Following the investigation, a heterogeneous direct competitive enzyme-linked immunosorbent assay (ELISA) format for microcystin-LR detection was developed, achieving a detection limit, LLD(80) = 0.022 μg L(-1). The system was then transferred to an affinity membrane sorbent-based ELISA. This was an amenable format for immunoassay incorporation into a disposable amperometric immunosensor device. This membrane-based ELISA achieved a detection limit, LLD(80) = 0.06 μg L(-1). A three-electrode immunosensor system was fabricated using thick-film screen-printing technology. Amperometric horseradish peroxidase transduction of hydrogen peroxide catalysis, at low reducing potentials, versus Ag/AgCl reference and carbon counter electrodes, was facilitated by hydroquinone-mediated electron transfer. A detection limit of 0.5 μg L(-1) for microcystin-LR was achieved. Similar levels of detection could be obtained using direct electrochemical sensing of the dye produced using the membrane-based ELISA. These techniques proved to be simple, cost-effective, and suitable for the detection of microcystin-LR in buffer and spiked tap and river water samples.

Topics: Benzothiazoles; Biosensing Techniques; Cross Reactions; Electrochemical Techniques; Enzyme-Linked Immunosorbent Assay; Immobilized Proteins; Immunoglobulin G; Marine Toxins; Membranes, Artificial; Microcystins; Peptides, Cyclic; Sulfonic Acids; Time Factors; Water Pollutants, Chemical

The estrogenic activity of cyanobacterial hepatotoxins microcystin-LR (MC-LR) and nodularin-R (NOD-R) was for the first time investigated invitro in a stably transfect cell line with an estrogen-regulated luciferase gene. Treatment of cells with NOD-R caused a dose-dependent increase in the luciferase activity. NOD-R gave rise to an induction of luciferase activity with an EC(50) value of 66.4 nM, whereas the positive control, 17beta-estradiol (E2) had an EC(50) of 9.6 pM. This indicates that NOD-R is a 6900-fold weaker inducer of luciferase than E2. In contrast, only a slight but significant activation of the luciferase gene was observed with MC-LR between 2.01 and 60.1 nM, and a maximal-induced response was observed with 10.1 nM, approximately 25% of the maximal effect obtained with 1 nM E2. The decrease in the luciferase activity at high MC-LR concentrations can be explained by a cytotoxic effect. No synergistic estrogenic effect was observed when each toxin was co-administrated with E2. However, the induction of the luciferase activity by NOD-R and MC-LR was inhibited by co-treatment with 1 microM of the pure estrogenic receptor (ER) antagonist ICI 182,780, thus proving the ER-dependency of the estrogenic effect.

Topics: Cell Line, Tumor; Cyanobacteria; Estradiol; Humans; Marine Toxins; Microcystins; Peptides, Cyclic; Reproducibility of Results

We report the esterification of the carboxyl groups of the cyclic peptide toxins nodularin-R and microcystin-LA to produce stable diacetoxymethyl and dipropionyloxymethyl ester derivatives. The derivatives had no activity but were reactivated upon esterase treatment. When injected into cells, the acyloxymethyl moieties were cleaved off and apoptosis induced. Linking the acyloxymethyl-ester moiety of these potent toxins to carriers destined for endocytosis paves the way for selective apoptosis induction in target (e.g., cancer) cells.

Topics: Animals; Apoptosis; Cytotoxins; Esterases; Esterification; Hepatocytes; Humans; Male; Mice; Microcystins; Peptides, Cyclic; Phenylglyoxal; Rats

This seven-year survey was primarily targeted to quantification of production of nodularin-R (NOD-R), a cyclic pentapeptide hepatotoxin, in Baltic Sea cyanobacteria waterblooms. Additionally, NOD-R and microcystin-LR (MC-LR; a cyclic heptapeptide toxin) sedimentation rates and NOD-R sediment storage were estimated. NOD-R production (70-2450 microg m(-3); approximately 1 kg km(-2) per season) and sedimentation rates (particles; 0.03-5.7 microg m(-2)d(-1); approximately 0.3kg km(-2) per season) were highly variable over space and time. Cell numbers of Nodularia spumigena did not correlate with NOD-R quantities. Dissolved NOD-R comprised 57-100% of total NOD-R in the predominantly senescent, low-intensity phytoplankton blooms and seston. Unprecedentedly intensive MC-LR sedimentation (0.56 microg m(-2)d(-1)) occurred in 2004. Hepatotoxin sedimentation rates highly exceeded those of anthropogenic xenobiotics. NOD-R storage in surficial sediments was 0.4-20 microg kg(-1) ( approximately 0.1 kg km(-2)). Loss of NOD-R within the chain consisting of phytoplankton, seston and soft sediments seemed very effective.

Topics: Bacterial Toxins; Cyanobacteria; Environmental Monitoring; Geologic Sediments; Marine Toxins; Microcystins; Nodularia; North Sea; Peptides, Cyclic; Seawater; Water Microbiology

Cyanobacteria are pathogenic prokaryotes and known for producing a high variety of cyclic hepatotoxic peptides in fresh and brackish water. Prominent members of these toxins are microcystin LR (MC LR) and nodularin (Nod), which are under suspicion to cause cancer. Various analytical methods have been reported for the detection of these cyclopeptides, and these are mainly based on liquid chromatography combined with mass spectrometric techniques. Here, we introduce a new approach based on the direct coupling of high-performance thin-layer chromatography (HPTLC) with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF MS) using the liquid matrix glycerol. The analysis of the cyclopeptides involves the application of three complementary methods: (i) HPTLC separation of MC LR and Nod, (ii) their detection and quantification by UV spectroscopy at lambda = 232 nm, and (iii) direct identification of separated analytes on the HPTLC plate by IR-MALDI-o-TOF MS. Calibration curves exhibited a linear relationship of amount of analyte applied for HPTLC and UV absorption (R(2) > 0.99). The limits of detection were 5 ng for UV spectroscopy and 3 ng for mass spectrometric analysis of individual peptides. This novel protocol greatly improves the sensitive determination of toxins from pathogenic cyanobacteria in complex water samples. It was successfully applied to the detection and quantification of MC LR and Nod in a spiked, processed environmental water sample.

Topics: Bacterial Toxins; Chromatography, Thin Layer; Cyanobacteria Toxins; Marine Toxins; Microcystins; Peptides, Cyclic; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrophotometry, Ultraviolet; Water Pollutants

Microcystins (MC) are cyanobacterial hepatotoxins responsible for animal-poisoning and human health incidents. Immunoassays provide a sensitive and fast means to detect these toxins, but cross-reactivity (CR) characteristic of different antibodies was variable. Here, we have produced and characterized a monoclonal antibody (Clone MC8C10) with highly specificity against the most frequent and most toxic variant of microcystins, MC-LR. MC8C10 is more specific against MC-LR among the reported antibodies before. The immunogen was synthesized from the modified MC-LR and bovine serum albumin (BSA). An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) with MC8C10 was established to detect the MCs in waters, which showed highly specificity with MC-LR, and have a detection limit for MC-LR 0.1 microg L(-1), the 50% inhibition concentration (IC50) for MC-LR was 1.8+/-0.1 microg L(-1) and the quantitative detection range was from 0.3 to 10 microg L(-1). The [4-arginine] microcystins and the nodularin-R showed lower cross-reactivates (CR<10%), and other MCs such as MC-LF and MC-LW are not recognized (CR<10(-4)). The analysis results of real water samples with ic-ELISA showed that all the coefficients of variation were less than 15%, and the recovery was (100.3+/-5.9)%. So the highly specific ic-ELISA will commendably suit for sensitive analysis for MC-LR in surface water as well as drinking water.

Topics: Animals; Antibodies, Monoclonal; Chromatography, High Pressure Liquid; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Hybridomas; Marine Toxins; Mice; Mice, Inbred BALB C; Microcystins; Peptides, Cyclic; Reproducibility of Results; Sensitivity and Specificity; Spleen; Water Pollutants

To assess the combinative role of aflatoxin B1(AFB1), cyanobacterial toxins (cyanotoxins), and hepatitis B virus (HBV) x gene in hepatotumorigenicity.. One-week-old animals carrying HBV x gene and their wild-type littermates were intraperitoneally (ip) injected with either single-dose AFB1 [6 mg/kg body weight (bw)], repeated-dose cyanotoxins (microcystin-LR or nodularin, 10 microg/kg bw once a week for 15 wk), DMSO (vehicle control) alone, or AFB1 followed by cyanotoxins a week later, and were sacrificed at 24 and 52 wk post-treatment.. AFB1 induced liver tumors in 13 of 29 (44.8%) transgenic mice at 52 wk post-treatment, significantly more frequent than in wild-type mice (13.3%). This significant difference was not shown in the 24-wk study. Compared with AFB1 exposure alone, MC-LR and nodularin yielded approximately 3-fold and 6-fold increases in the incidence of AFB(1)-induced liver tumors in wild-type animals at 24 wk, respectively. HBV x gene did not further elevate the risk associated with co-exposure to AFB1 and cyanotoxins. With the exception of an MC-LR-dosed wild-type mouse, no liver tumor was observed in mice treated with cyanotoxins alone at 24 wk. Neither DMSO-treated transgenic mice nor their wild-type littermates had pathologic alterations relevant to hepatotumorigenesis in even up to 52 wk.. HBV x gene and nodularin promote the development of AFB(1)-induced liver tumors. Co-exposure to AFB1 and MC-LR tends to elevate the risk of liver tumors at 24 wk relative to exposure to one of them. The combinative effect of AFB1, cyanotoxins and HBVx on hepatotumorigenesis is weak at 24 wk.

Topics: Aflatoxin B1; Animals; Bacterial Toxins; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cyanobacteria Toxins; Dose-Response Relationship, Drug; Drug Synergism; Liver Neoplasms; Male; Marine Toxins; Mice; Mice, Transgenic; Microcystins; Peptides, Cyclic; Poisons; Polymerase Chain Reaction; Time Factors; Trans-Activators; Viral Regulatory and Accessory Proteins

Topics: Bacterial Toxins; Carcinogens; Cyanobacteria Toxins; Humans; Marine Toxins; Microcystins; Nitrates; Nitrites; Peptides, Cyclic

Depending on the class of microcystin the protein phosphatase inhibition assay shows different sensitivities to different classes of toxin. We have determined that the IC50 values obtained from dose-response curves for the inhibition of the enzyme by micro-cystin LR, nodularin, YR, and RR were 2.2, 1.8, 9 and 175 nM, respectively. When equimolar amounts of these toxins were determined by the ELISA assay with microcystin LR as the standard, the assay showed equivalence in toxin responses. However, when the toxins were determined by the protein phosphatase inhibition assay using microcystin LR as the standard, the ratios of the values determined by PP-2A to ELISA decreased in the order: nodularin (2.23) microcystin LR (1.1)> microcystin YR (0.63)> microcystin RR (0.06). When the ratios for each standard were plotted against the IC50 values, the log-log plot was negative linear, and the lowest value for the IC50 corresponded with the lowest ratio. The differential sensitivity of the PP-2A assay to the various standards was used to establish an indicative toxicity ranking (ITR) where a ranking of 1 (the highest) was assigned to ratios of > or = 0.8 or greater, and 3 (the lowest) to values < or = 0.2. The three ranking classes corresponded to toxin equivalence represented by the four standards. The new method allows not only the determination of microcystin toxins in terms of stoichiometry (ELISA) but also in terms of indicative toxicity. The method can be performed using the same instrument (e.g. multiwell fluorimeter with absorbance capability) and offers an advantage to methods presently used to determine microcystins (e.g. ELISA or LC-MS). The former has the propensity to overestimate toxicity because it measures equivalence to microcystin LR and is a stoichiometric measurement and the latter has the disadvantage in that relatively few of the microcystins that occur naturally are available as standards. The new method was applied to the analysis of sample from lakes and streams from temperate locations and to extracts of cyanobacterial mats from ponds and streams in cold temperature locations.

Topics: Bacterial Toxins; Chromatography, Liquid; Cyanobacteria; Enzyme-Linked Immunosorbent Assay; Marine Toxins; Mass Spectrometry; Microcystins; Peptides, Cyclic; Phosphoprotein Phosphatases; Water Pollutants

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.

Topics: Bacterial Toxins; Biodegradation, Environmental; Chromatography, High Pressure Liquid; Cyanobacteria; Enzyme Inhibitors; Marine Toxins; Microcystins; Peptides, Cyclic; Spectrometry, Mass, Electrospray Ionization

Microcystin-LR (MCYST-LR) and nodularin (NOD) are known as tumor promoters in experimental animals and so present potential health threats for humans. Although their hepatotoxic mechanisms have been very well documented, many other effects of these toxins are relatively undescribed, indeed controversial, notably those related to their genotoxicity. In the present investigation, we examined how these toxins could induce DNA damage using a combination of in vitro and in vivo approaches. We first used the (32)P-postlabeling assay to test hydrophobic adduct formation on DNA from primary cultured rat hepatocytes treated with noncytotoxic concentrations of MCYST-LR and NOD (2 and 10 ng/mL). Analysis of the autoradiograms of DNA digests isolated from the hepatocytes did not show any hydrophobic DNA adduct formation. However, these toxins significantly decreased the amount of hydrophobic endogenous adducts, termed I compounds. We next investigated oxidative DNA damage by using the (32)P-postlabeling assay to analyze 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) content as a biomarker of possible DNA lesions. Both MCYST-LR and NOD significantly enhanced 8-oxo-dG in time- and dose-dependent manner in vitro in primary cultured hepatocytes and in vivo in rat liver cells. Thus, it appears that the depletion of endogenous DNA adducts (I compounds) and/or the increase of 8-oxo-dG levels by MCYST-LR and NOD could be involved in the formation of hepatic tumors during long-term exposure to these cyanobacterial hepatotoxins.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Biomarkers; Cell Culture Techniques; Deoxyguanosine; DNA Damage; Enzyme Inhibitors; Hepatocytes; Liver; Liver Neoplasms; Male; Marine Toxins; Microcystins; Oxidative Stress; Peptides, Cyclic; Rats; Rats, Sprague-Dawley

Cyanobacterial toxins have been shown to have a far-reaching impact-from aquatic organisms to human health. Aquatic organisms are typically exposed in their natural environment to toxic cyanobacteria, and exposure can occur via ingestion of cyanobacterial cells or by bioaccumulation of water-borne toxin. The aquaculture and fisheries of crustaceans are among the most important seafood industries. Concomitant with the growth of this industry, the importance of the health of crustaceans increased. The black tiger prawn is the major cultivated prawn in Australia. The aquaculture of these prawns takes place in shallow ponds, where blooms, often of cyanobacteria, develop. Cyanobacterial toxins were hypothesized to contribute to the mortality of prawns. Many aquatic organisms have the possibility of detoxifying cyanobacterial toxins via conjugation to glutathione. The presence of several classes of the cytosolic glutathione S-transferase system in black tiger prawns-mu, pi, theta, alpha, and tau-was shown using different substrates for measurement. Injection experiments with microcystin-LR and feeding experiments with nodularin revealed elevation of GST activity in different types of prawn tissue in parallel with reduction in the GST classes. Correlation analyses of toxin content of the prawns with GST activity showed that low toxin content was correlated with high elevation of enzymes and high toxin content with low elevation of enzymes.

Topics: Animals; Aquaculture; Enzyme Inhibitors; Glutathione Transferase; Marine Toxins; Microcystins; Microsomes; Penaeidae; Peptides, Cyclic

Microcystin-LR (MCYST-LR) and nodularin (NOD) produced by cyanobacteria are potent and specific hepatotoxins. The induction of free-radical formation, reduction of glutathione levels and induction of DNA damage are three major events found in rat hepatocytes treated with these hepatotoxins. However, the mechanism of MCYST-LR- and NOD-mediated induction of oxidative DNA damage has not been fully elucidated. The objective of this study was to determine whether MCYST-LR and NOD increase the formation of a DNA oxidative damage marker such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in vitro in primary rat hepatocytes and in vivo in rat liver cells. Rat hepatocytes were exposed to MCYST-LR or NOD at low doses (2 and 10 ng/ml), at which there is no evidence of morphologically apparent cytotoxic effects, as well as an induced dose- and time-dependent formation of 8-oxo-dG. Moreover, MCYST-LR treatment of rats (50 microg/kg, ip) resulted in a significant increase of 8-oxo-dG in liver DNA, at 24 h after treatment before decreasing at 48 h. However, NOD-induced DNA damage was increased both at 24 and 48 h, in contrast to the MCYST-LR-induced effect. The effects on this oxidative DNA damage marker indicates that MCYST-LR and NOD do evoke oxidative stress, which may contribute, at least in part, to their liver toxicity and carcinogenicity during long-term exposure.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Biomarkers; Cells, Cultured; Deoxyguanosine; DNA; DNA Damage; Enzyme Inhibitors; Hepatocytes; Humans; Liver; Male; Marine Toxins; Microcystins; Peptides, Cyclic; Phosphorus Radioisotopes; Rats; Rats, Sprague-Dawley

The toxicity and metabolism of the cyanobacterial toxins microcystin-LR (MCLR), Dhb-microcystin-HtyR and nodularin were investigated in the cysts, nauplii and adults of the brine shrimp Artemia salina. The presence of the phase II detoxication system glutathione S-transferase (sGST) in these stages was shown using different substrates. Exposure of adult A. salina to the toxins led to an elevation of GST activity in vivo. All three toxins were conjugated to glutathione via GST, which has been shown as an initial step of microcystin and nodularin detoxication.

Topics: Animals; Artemia; Bacterial Toxins; Cyanobacteria; Enzyme Inhibitors; Glutathione; Glutathione Transferase; Inactivation, Metabolic; Marine Toxins; Microcystins; Peptides, Cyclic

Bank filtration offers a cost effective and low maintenance technique for the removal of cyanobacterial hepatotoxins from drinking water. For bank filtration to be effective, the toxins must be degraded. The broad aim of this research was to determine whether the hepatotoxins, nodularin and microcystin-LR, could be completely removed from the soil/water matrix of three soils by microbial degradation. The results indicated that complete toxin removal was possible within 10-16 d in 2/3 soils that were incubated in the dark at 20 degrees C. The soils with the highest organic carbon content (2.9%) and the highest clay content (16.1%) were the most effective at removing the toxins in batch experiments. However, the sandy soil (98.5% sand) was incapable of degrading either toxin. The half-lives of toxin losses due to adsorption, desorption and degradation were calculated and for all soils. The degradation process had the highest half-life for both toxins. This suggested that degradation was likely to be the rate-limiting step of complete toxin removal. It was concluded that when a bank filtration site was being chosen, the degradation potential and the textural properties of the riverbank soil would be important when considering complete removal of cyanobacterial hepatotoxins.

Topics: Adsorption; Cyanobacteria; Filtration; Half-Life; Marine Toxins; Microcystins; Peptides, Cyclic; Silicon Dioxide; Soil; Soil Microbiology; Water Microbiology; Water Supply

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-Asp(3),(E)-Dhb(7)]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 LC(50) values [D-Asp(3),(E)-Dhb(7)]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 LC(50) 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-Asp(3),(E)-Dhb(7)]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-Asp(3),(E)-Dhb(7)]microcystin-RR when compared to the N-methyldehydroalanine-containing microcystins.

Topics: Animals; Bacterial Toxins; Biological Assay; Cyanobacteria; Cyanobacteria Toxins; Decapoda; Dose-Response Relationship, Drug; Gas Chromatography-Mass Spectrometry; Lethal Dose 50; Magnetic Resonance Spectroscopy; Marine Toxins; Microcystins; Peptides, Cyclic

Cyanobacteria (blue-green algae) (e.g., Microcystis and Nodularia spp.) capable of producing toxic peptides are found in fresh and brackish water worldwide. These toxins include the microcystin (MC) heptapeptides (>60 congeners) and the nodularin pentapeptides (ca. 5 congeners). Cyanobacterial cyclic peptide toxins are harmful to man, other mammals, birds, and fish. Acute exposure to high concentrations of these toxins causes liver damage, while subchronic or chronic exposure may promote liver tumor formation. The detection of cyclic peptide cyanobacterial toxins in surface and drinking waters has been hampered by the low limits of detection required and that the present routine detection is restricted to a few of the congeners. The unusual beta-amino acid ADDA (4E,6E-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) is present in most (>80%) of the known toxic penta- and heptapeptide toxin congeners. Here, we report the synthesis of two ADDA-haptens, the raising of antibodies to ADDA, and the development of a competitive indirect ELISA for the detection of microcystins and nodularins utilizing these antibodies. The assay has a limit of quantitation of 0.02-0.07 ng/mL (depending on which congeners are present), lower than the WHO-proposed guideline (1 ng/mL) for drinking water, irrespective of the sample matrix (raw water, drinking water, or pure toxin in PBS). This new ELISA is robust, can be performed without sample preconcentration, detects toxins in freshwater samples at lower concentrations than does the protein phosphatase inhibition assay, and shows very good cross-reactivity with all cyanobacterial cyclic peptide toxin congeners tested to date (MC-LR, -RR, -YR, -LW, -LF, 3-desmethyl-MC-LR, 3-desmethyl-MC-RR, and nodularin).

Topics: Cyanobacteria; Immunoassay; Marine Toxins; Microcystins; Peptides, Cyclic; Water Pollutants; Water Pollution

An ultrasensitive assay is described for microcystin-LR and other substances (microcystins, nodularin, okadaic acid, calyculin A, tautomycin) which block the active site of protein phosphatases (PP) 1 and 2A. The assay is based on competition between the unknown sample and [125I]microcystin-YR for binding to the catalytic subunit of PP2A. The PP2A-bound [125I]microcystin-YR was stable (half-time of dissociation = 1.8 h), allowing non-bound [125I]microcystin-YR to be removed by Sephadex G-50 size-exclusion chromatography. Compared to current assays based on inhibition of protein phosphatase activity the present assay was more robust against interference (from fluoride, ATP, histone, and casein), and had an even better sensitivity. The detection limit was below 50 pM (2.5 fmol) for nodularin and microcystin-LR, and below 200 pM (10 fmol) for okadaic acid. The method was used successfully to detect extremely low concentrations of either microcystin or nodularin in drinking water or seawater, and okadaic acid in shellfish extract.

Topics: Bacterial Toxins; Binding Sites; Binding, Competitive; Biological Assay; Iodine; Marine Toxins; Microcystins; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Seawater; Shellfish; Time Factors; Tissue Extracts; Toxins, Biological; Water Supply

The preparation of antibodies against the liver toxin microcystin, as described here, is of major importance for its detection and purification in food and water, and for a therapeutic approach to neutralize the toxin by passive immunization. Microcystin-LR (MLR) and microcystin-RR (MRR) were purified from cyanobacterial cell materials by extraction, Sephadex LH-20-, ODS silica gel-, ionic exchange and RP-HPLC-chromatography. In order to reduce the toxicity for parenteral administration, microcystins were coupled by the carbodiimide method to poly-L-lysine (PLL(50.000)). Mice and rabbits were immunized with the conjugates in the presence of two lipopeptide immunoadjuvants (P(3)CSK(4) and P(3)CS-T(h)). High MLR-specific antibody levels were observed after parenteral coadministration of antigen and lipopeptides, whereas no anti-MLR antibodies were obtained with free microcystin or the microcystin-PLL(50.000)-conjugate in the absence of lipopeptide. In oral immunization, coadministration of antigen and adjuvants resulted in an accelerated development of anti MLR-specific antibodies and high antibody levels. Using the antisera, we could detect different microcystins and nodularin down to a concentration range of 10-50 ng/ml by a competitive inhibition ELISA; detection of microcystins in crude cell preparations was also possible. Furthermore, microcystins from different sources could be detected and discriminated from cyclic cyanopeptolines.

Topics: Administration, Oral; Animals; Antibody Formation; Enzyme-Linked Immunosorbent Assay; Female; Immune Sera; Immunization; Marine Toxins; Mice; Mice, Inbred BALB C; Microcystins; Peptides, Cyclic; Rabbits

The catalytic cores of PP-1c and PP-2B (calcineurin) are structurally conserved. However, PP-2B is resistant to inhibition by toxins of the okadaic acid and cyclic peptide classes, while PP-1c is potently inhibited. Molecular docking of the structure of microcystin-LR onto the catalytic core of PP-2B identified residues that may be responsible for blocking access of toxins to the catalytic site. Amino acids in PP-1c were substituted with these PP-2B residues to investigate their contribution to PP-2B toxin resistance. Mutants of PP-1c were also produced to test the importance of hydrophobic interactions to toxin binding. Our results suggest that different classes of toxin inhibitors interact with the same hydrophobic side chains of PP-1c through different mechanisms. Substitution of amino acids in PP-1c with PP-2B residues demonstrated no highly significant changes in toxin inhibition. We hypothesize that an interaction outside the catalytic core causing the L7 loop of PP-2B to block the catalytic site may be responsible for PP-2B resistance to toxins.

Topics: Animals; Binding Sites; Calcineurin; Catalytic Domain; Cattle; Crystallography, X-Ray; Escherichia coli; Humans; Intracellular Signaling Peptides and Proteins; Marine Toxins; Microcystins; Models, Molecular; Mutagenesis, Site-Directed; Okadaic Acid; Peptides, Cyclic; Protein Conformation; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Rats; Recombinant Proteins; Toxins, Biological

Cyanobacteria are known to produce hepatotoxic substances, the functional and ecological role of these toxins, however, remains largely unclear. Toxic properties of cyanobacteria collected in Antarctica were investigated to determine whether toxin-producing species can also be found under these environmental conditions. Samples were collected from meltwater ponds on the McMurdo Ice Shelf, Antarctica in the summers of 1997 to 1999. These ponds are colonized by benthic algae and cyanobacterial mats. Oscillatoriales, Nodularia sp., and Nostoc sp. constituted the major taxa in freshwater ponds, while Nostoc sp. was missing from brackish and saline ponds. Samples were taken from either floating, submerged or benthic mats, and extracted for in vitro toxicity testing. The presence of toxins was determined by the phosphatase-inhibition assay and by high performance liquid chromatography. The cytotoxic properties of the extracts were investigated in hepatocytes determining 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide metabolism and trypan blue dye exclusion. The results show that all cyanobacterial extracts display phosphatase-inhibiting activity, of which approximately half had significantly greater than 50% inhibiting activity. The presence of nodularin and microcystin-LR was established by high performance liquid chromatography. Cytotoxic properties, independent of the phosphatase inhibiting activity, were also detected. Toxic strains of cyanobacteria can therefore also be found in Antarctica and this finding may lead to further insight into potential ecological roles of cyanobacterial phosphatase inhibiting toxins.

Topics: Animals; Antarctic Regions; Bacterial Toxins; Biomass; Chromatography, High Pressure Liquid; Cyanobacteria; Enzyme Inhibitors; Fresh Water; Liver; Marine Toxins; Microcystins; Oncorhynchus mykiss; Peptides, Cyclic; Phosphoric Monoester Hydrolases; Tetrazolium Salts; Thiazoles; Trypan Blue

The protein phosphatase (PP) inhibitors nodularin and microcystin-LR induced apoptosis with unprecedented rapidity, more than 50% of primary hepatocytes showing extensive surface budding and shrinkage of cytoplasm and nucleoplasm within 2 min. The apoptosis was retarded by the general caspase inhibitor Z-VAD.fmk. To circumvent the inefficient uptake of microcystin and nodularin into nonhepatocytes, toxins were microinjected into 293 cells, Swiss 3T3 fibroblasts, promyelocytic IPC-81 cells, and NRK cells. All cells started to undergo budding typical of apoptosis within 0.5 - 3 min after injection. This was accompanied by cytoplasmic and nuclear shrinkage and externalization of phosphatidylserine. Overexpression of Bcl-2 did not delay apoptosis. Apoptosis induction was slower and Z-VAD.fmk independent in caspase-3 deficient MCF-7 cells. MCF-7 cells stably transfected with caspase-3 showed a more rapid and Z-VAD.fmk dependent apoptotic response to nodularin. Rapid apoptosis induction required inhibition of both PP1 and PP2A, and the apoptosis was preceded by increased phosphorylation of several proteins, including myosin light chain. The protein phosphorylation occurred even in the presence of apoptosis-blocking concentrations of Z-VAD.fmk, indicating that it occurred upstream of caspase activation. It is suggested that phosphatase-inhibiting toxins can induce caspase-3 dependent apoptosis in an ultrarapid manner by altering protein phosphorylation.

Topics: 3T3 Cells; Animals; Antifungal Agents; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cell Line; Cell Line, Transformed; Enzyme Inhibitors; Gene Expression; Humans; Intracellular Fluid; Marine Toxins; Mice; Microcystins; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Pyrans; Rats; Rats, Wistar; Spiro Compounds; Tumor Cells, Cultured

A number of algal toxins were tested for the ability to induce apoptosis (regulated cell death) in primary hepatocytes from salmon and rat. The tested toxins included the liver targeting substances microcystin-LR and nodularin, substances associated with the diarrhetic shellfish poison complex (okadaic acid, dinophysistoxin-1 and pectenotoxin-1) and calyculin A. All toxins induced apoptosis in both salmon and rat hepatocytes in less than 2 h. The apoptotic changes were evident both by electron and light microscopy and were counteracted by the caspase inhibitor ZVAD-fmk and by the Ca2+/calmodulin dependent kinase II inhibitor KN-93. The salmon hepatocytes were 10-20-fold more sensitive to okadaic acid and dinophysistoxin-1 (EC50=20 nM) than rat hepatocytes and other mammalian cell lines tested. An assay was devised using hepatocyte apoptosis as parameter for detection of algal toxins. This assay was at least as sensitive as HPLC determination for okadaic acid in mussel extracts. It also detected algal toxins which do not inhibit protein phosphatases, like pectenotoxin-1. Subapoptotic concentrations of the toxins inhibited hepatocyte aggregation. Using this parameter, less than 200 pg okadaic acid could be detected. In conclusion, salmon hepatocytes in suspension culture provide a rapid and sensitive system for detection of a broad range of apoptogenic toxins.

Topics: Animals; Apoptosis; Cell Aggregation; Cells, Cultured; Eukaryota; Liver; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Peptides, Cyclic; Pyrans; Rats

Protein phosphatases play significant roles in signal transduction pathways pertaining to cell proliferation, gene expression, and neurotransmission. Serine/threonine phosphatases PP1 and PP2A, which are closely related in primary structure (approximately 50%), are inhibited by a structurally diverse group of natural toxins. As part of our study toward understanding the mechanism of inhibition displayed by these toxins, we have developed research in two directions: (1) The standardization of an assay to be used in acquisition of the structure--activity relationship of inhibition data is reported. This nonradioactive assay affords detection levels of molecular phosphate released from a phosphorylated hexapeptide in subnanomolar quantities. The comparison of our IC50 values of these inhibitors against corresponding literature data provided validation for our method. (2) Computational analysis provided a global model for binding of these inhibitors to PP1. The natural toxins were shown to possess remarkably similar three-dimensional motifs upon superimposition and van der Waals minimization within the PP1 active site.

Topics: Antifungal Agents; Crystallography, X-Ray; Enzyme Inhibitors; Marine Toxins; Microcystins; Models, Molecular; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Binding; Protein Conformation; Pyrans; Signal Transduction; Spiro Compounds; Stereoisomerism; Structure-Activity Relationship; Toxins, Biological

Nodularin is a new liver carcinogen possessing a potent tumor-promoting activity in rat liver, mediated through inhibition of protein phosphatases 1 and 2A, and a weak initiating activity. Since we previously reported evidence that nodularin up-regulated expression of the tumor necrosis factor alpha gene (TNF alpha) and early-response genes in rat liver after its i.p. administration, and since TNF alpha had tumor-promoting activity in vitro, it is possible that TNF alpha itself is involved in liver tumor promotion. We investigated whether hepatocytes themselves induce expression of the TNF alpha gene and early-response genes in primary cultured rat hepatocytes treated with nodularin. Like nodularin, microcystin-LR, which is another liver tumor promoter belonging to the okadaic acid class, strongly induced TNF alpha gene expression in rat hepatocytes, as well as TNF alpha release from those cells into the medium. On the other hand, 12-O-tetradecanoylphorbol-13-acetate, which has been reported to induce no tumor promotion in rat liver, induced no apparent expression of the TNF alpha gene in primary cultured rat hepatocytes. As for the expression of early-response genes, 1 microM nodularin or microcystin-LR induced expression of the c-jun, jun B, jun D, c-fos, fos B and fra-1 genes in the hepatocytes, and the expression of these genes was prolonged up to 24 h, suggesting mRNA stabilization induced by inhibition of protein phosphatases 1 and 2A. This paper presents new evidence that the TNF alpha gene and early-response genes were expressed in hepatocytes treated with a liver tumor promoter.

Topics: Animals; Carcinogens; Cells, Cultured; Enzyme Inhibitors; Gene Expression; Genes, fos; Genes, Immediate-Early; Genes, jun; Liver; Liver Neoplasms, Experimental; Male; Marine Toxins; Microcystins; Peptides, Cyclic; Rats; Rats, Inbred F344; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Heptapeptide microcystin and pentapeptide motuporin (nodularin-V) are equipotent inhibitors of type-1 and type-2A protein phosphatase catalytic subunits (PP-1c and PP-2Ac). Herein we describe elucidation of the molecular mechanisms involved in the interaction of these structurally similar hepatotoxins with PP-1c/PP-2Ac and identification of an important functional difference between their mode of interaction with these enzymes. Microcystin-LR, microcystin-LA, and microcystin-LL were found to interact with PP-2Ac and PP-1c by a two-step mechanism involving rapid binding and inactivation of the protein phosphatase (PPase) catalytic subunit, followed by a slower covalent interaction (within hours). Covalent adducts comprising PPase-toxin complexes were separated from free PPase by C-18 reverse-phase liquid chromatography, thus allowing the time course of covalent adduct formation to be quantitated. In contrast to microcystins, motuporin (nodularin-V) and nodularin-R were unable to form covalent complexes with either PP-1c or PP-2Ac even after 96 h incubation. Specific reduction of microcystin-LA to dihydromicrocystin-LA abolished the ability of the toxin to form a covalent adduct with PP-2Ac. Specific methyl esterification of the single Glu residue in microcystin-LR rendered this toxin inactive as a PPase inhibitor and abolished subsequent formation of a covalent adduct. Our data indicate that inactivation of PP-2Ac/PP-1c by microcystins precedes covalent modification of the PPases via a Michael addition reaction between a nucleophilic phosphatase residue and Mdha in the heptapeptide toxin. In contrast, following rapid inactivation of PP-2Ac/PP-1c by motuporin, the equivalent N-methyldehydrobutyrine residue in this toxin is unreactive and does not form a covalent bond with the PPases. These results are consistent with structural data for (i) the NMR solution structures of microcystin-LR and motuporin, which indicate a striking difference in the relative positions of their corresponding dehydroamino acids in the toxin peptide backbone, and (ii) X-ray crystallographic data on an inactive complex between PP-1c and microcystin-LR, which show a covalent bond between Cys-273 and the bound toxin.

Topics: Enzyme Inhibitors; Kinetics; Marine Toxins; Microcystins; Peptides, Cyclic; Phosphoprotein Phosphatases

A comparison of the structures of two cyanobacterial toxins yields insights into how they may inhibit protein phosphatase-1 and -2A and why microcystins but not motuporin may covalently modify their protein phosphatase targets.

Topics: Amino Acid Sequence; Magnetic Resonance Spectroscopy; Marine Toxins; Microcystins; Models, Molecular; Molecular Sequence Data; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Binding; Protein Phosphatase 1; Protein Structure, Tertiary; Solutions

Nodularin and microcystin-LR are cyanobacterial toxins and environmental hazards. Nodularin inhibits protein phosphatases 1 and 2A with the same potency as does microcystin-LR, which has recently been identified as a potent tumor promoter in rat liver. Our results suggested that nodularin is also a new tumor promoter in rat liver. A two-stage carcinogenesis experiment in rat liver initiated with diethylnitrosamine and without partial hepatectomy revealed that nodularin stimulated glutathione S-transferase placental form-positive foci in rat liver more effectively than did microcystin-LR, and that nodularin alone induced glutathione S-transferase placental form-positive foci as well as did diethylnitrosamine alone. Thus, nodularin itself is a new liver carcinogen, and microcystin-LR is a tumor promoter rather than a carcinogen. Nodularin induced hyperphosphorylation of cytokeratin peptides 8 and 18 in primary cultured rat hepatocytes 20% more effectively than did microcystin-LR, suggesting that nodularin penetrates more easily into the hepatocytes than does microcystin-LR. Nodularin up-regulated induction of c-jun, jun-B,jun-D,c-fos,fos-B, and fra-1 mRNA transcripts in rat liver after i.p. administration, and the accumulation of the mRNA transcripts was sustained for over 9 h after treatment. The environmental hazards of cyanobacterial toxins are discussed in relation to human primary liver cancer in Qidong county in the People's Republic of China. Our results support this hypothesis and indicate the need for prevention measures against cyanobacterial toxins.

Topics: Animals; Bacterial Toxins; Carcinogens; Diethylnitrosamine; Immediate-Early Proteins; Keratins; Liver; Liver Neoplasms, Experimental; Male; Marine Toxins; Microcystins; Peptides, Cyclic; Phosphorylation; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Rats, Inbred F344; RNA, Messenger

A chimeric mutant was constructed in which a 4-amino acid region (GEFD, residues 274-277) of rabbit muscle protein phosphatase-1 was replaced with the sequence YRCG corresponding to residues 267-270 of rabbit protein phosphatase-2A. This was based on the findings of a gene mutation in okadaic acid-resistant cells which results in a Cys-->Gly conversion in protein phosphatase-2A. The YRCG mutant of protein phosphatase-1 was expressed and purified. The properties of the mutant enzyme were investigated in terms of its sensitivity toward several toxin inhibitors (okadaic acid, microcystin, nodularin, calyculin A, and cantharidic acid), as well as inhibitor-2. The mutant enzyme exhibited a gain of function in the form of a 10-fold increased sensitivity toward okadaic acid that suggests this region is involved in toxin binding. Significant changes in sensitivity to inhibitor-2 and several of the other toxins were also observed, indicating that these may have a common binding region.

Topics: Amino Acid Sequence; Animals; DNA Primers; Ethers, Cyclic; Kinetics; Marine Toxins; Microcystins; Molecular Sequence Data; Mutagenesis, Site-Directed; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Phosphatase 2; Rabbits; Recombinant Fusion Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship

Recent studies suggest that the ability to inhibit the activity of certain serine/threonine protein phosphatases underlies the toxicity of several natural compounds including: okadaic acid, microcystin-LR, nodularin, calyculin A and tautomycin. To characterize further the actions of these toxins, this study compares the inhibitory effects of okadaic acid, chemical derivatives of okadaic acid, microcystin-LR, microcystin-LA, nodularin, calyculin A and tautomycin on the activity of serine/threonine protein phosphatases types 1 (PP1), 2A (PP2A) and a recently identified protein phosphatase purified from bovine brain (PP3). This study shows that, like PP1 and PP2A, the activity of PP3 is potently inhibited by okadaic acid, both microcystins, nodularin, calyculin A and tautomycin. Further characterization of the toxins employing the purified catalytic subunits of PP1, PP2A and PP3 under identical experimental conditions indicates that: (a) okadaic acid, microcystin-LR, and microcystin-LA inhibit PP2A and PP3 more potently than PP1 (order of potency PP2A > PP3 > PP1); (b) nodularin inhibits PP1 and PP3 at a similar concentration that is slightly higher than that which affects PP2A, and (c) both calyculin A and tautomycin show little selectivity among the phosphatases tested. This study also shows that the chemical modification of the (C1) carboxyl group of okadaic acid can have a profound influence on the inhibitory activity of this toxin. Esterification of okadaic acid, producing methyl okadaate, or reduction, producing okadaol, greatly decreases the inhibitory effects against all three enzymes tested. Further reduction, producing 1-nor-okadaone, or acetylation, producing okadaic acid tetraacetate, results in compounds with no inhibitory activity. In contrast, the substitution of alanine (-LA) for arginine (-LR) in microcystin has no apparent effect on the inhibitory activity against PP1, PP2A or PP3.

Topics: Animals; Antifungal Agents; Cattle; Cyclic AMP-Dependent Protein Kinases; Esterification; Ethers, Cyclic; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Oxidation-Reduction; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylase b; Phosphorylase Kinase; Pyrans; Rabbits; Spiro Compounds

This study investigates the occurrence and regulation of serine/threonine protein phosphatases (PPases) in insulin-secreting RINm5F insulinoma cells. PPases types 1 and 2A were identified in crude RINm5F cell homogenates by both enzymatic assay and Western blot analysis. We then characterized and compared the inhibitory actions of several compounds isolated from cyanobacteria, marine dinoflagellates and marine sponges, (viz. okadaic acid, microcystin-LR, calyculin-A and nodularin) cation-independent PPase activities in RINm5F cell homogenates. It was found that okadaic acid was the least potent inhibitor (IC50 approximately 10(-9) M, IC100 approximately 10(-6) M), while the other compounds exhibited IC50 values of approximately 5 x 10(-10) M and IC100 approximately 5 x 10(-9) M. The findings indicate that the inhibitory substances employed in this study may be used pharmacologically to investigate the role of serine/threonine PPases in RINm5F cell insulin secretion, a process that is likely to be regulated to a major extent by protein phosphorylation.

Topics: Animals; Ethers, Cyclic; In Vitro Techniques; Insulinoma; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Pancreatic Neoplasms; Peptides, Cyclic; Phosphoprotein Phosphatases; Rabbits; Rats; Tumor Cells, Cultured

The 3-dimensional structures of two cyanobacterial hepatotoxins microcystin-LR, a cyclic heptapeptide and nodularin, a cyclic pentapeptide, and the novel amino acid ADDA (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid) were constructed, and optimized using the CHEM-X molecular mechanics program. The peptide rings were planar and of rectangular shape. Optimized ADDA formed a U-shape and a difference in the orientation of ADDA with respect to the peptide ring of the two hepatotoxins was observed.

Topics: Computer Simulation; Cyanobacteria; Liver; Marine Toxins; Microcystins; Models, Chemical; Peptides, Cyclic; Protein Conformation

An isolation procedure was developed to provide within one day microcystin-LR, a cyclic heptapeptide toxin from Microcystis aeruginosa PCC 7806. After ODS (octadecylsilyl) solid phase extraction, the crude toxin fraction was chromatographed using a strong anion exchange column. The toxin was eluted with 0.02 M ammonium bicarbonate. An at least 95% purity was revealed on HPLC separation by monitoring at 214 nm. Application of the procedure to the cyclic pentapeptide toxin nodularin from Nodularia spumigena AV2 was examined.

Topics: Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cyanobacteria; Marine Toxins; Microcystins; Peptides, Cyclic

Topics: Chromatography, High Pressure Liquid; Cyanobacteria; Marine Toxins; Microcystins; Peptides, Cyclic

Three microcystins, YR, LR and RR and nodularin, all of which are hepatotoxic compounds, inhibited dose-dependently the activity of protein phosphatase 2A in and the specific [3H]okadaic acid binding to a cytosolic fraction of mouse skin, as strongly as okadaic acid. However, microcytins and nodularin did not induce any effects on mouse skin or primary human fibroblasts. Microinjection of microcystin YR into primary human fibroblasts induced morphological changes which were induced by incubation with okadaic acid. Microcystins and nodularin penetrate into the epithelial cells of mouse skin and human fibroblasts with difficulty, which reflects tissue specificity of the compounds.

Topics: Animals; Cells, Cultured; Cytosol; Ethers, Cyclic; Fibroblasts; Humans; Marine Toxins; Mice; Microcystins; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Plants, Toxic; Protein Binding; Protein Phosphatase 2; Skin

Microcystins and nodularin, isolated from toxic blue-green algae, are hepatotoxic monocyclic polypeptides. Both microcystins and nodularin inhibited in vitro protein phosphatase activity present in a cytosolic fraction of mouse liver, bound to the okadaic acid receptors, protein phosphatases 1 and 2A, and thus resulted in the increase of phosphoproteins; this was referred to as the apparent "activation" of protein kinases. Their concentrations causing 50% of the maximal effects are comparable to that of okadaic acid, a potent protein phosphatase inhibitor and a potent tumor promoter, in the nanomolar range of concentration. The increase of phosphoproteins was observed in rat primary cultured hepatocytes and was subsequently associated with morphological changes, which appeared to be a step in the process of hepatotoxicity. The well-known hepatotoxic compounds, alpha-amanitin and phalloidin, did not show any effects similar to those of microcystins, nodularin and okadaic acid. It is suggested that the hepatotoxicity of microcystins and nodularin may result from inhibition of protein phosphatases and the increase of phosphoproteins.

Topics: Animals; Cells, Cultured; Cyanobacteria; Ethers, Cyclic; Liver; Male; Marine Toxins; Mice; Microcystins; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphoproteins; Protein Kinases; Rats; Rats, Inbred Strains

The cyclic peptide hepatotoxins microcystin-LR, 7-desmethyl-microcystin-RR and nodularin are potent inhibitors of the protein phosphatases type 1 and type 2A. Their potency of inhibition resembles calyculin-A and to a lesser extent okadaic acid. These hepatotoxins increase the overall level of protein phosphorylation in hepatocytes. Evidence is presented to indicate that in hepatocytes the morphological changes and effects on the cytoskeleton are due to phosphatase inhibition. The potency of these compounds in inducing hepatocyte deformation is similar to their potency in inhibiting phosphatase activity. These results suggest that the hepatotoxicity of these peptides is related to inhibition of phosphatases, and further indicate the importance of the protein phosphorylation in maintenance of structural and homeostatic integrity in these cells.

Topics: Amino Acid Sequence; Animals; Carrier Proteins; Chickens; Cyanobacteria; Enzyme Inhibitors; Intracellular Signaling Peptides and Proteins; Liver; Marine Toxins; Microcystins; Molecular Sequence Data; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Proteins