Cyanobacterial Toxin microcystin-LR Research Articles

Effects of Cyanobacterial Harmful Algal Bloom Toxin Microcystin-LR on Gonadotropin-Dependent Ovarian Follicle Maturation and Ovulation in Mice.

Cyanobacterial harmful algal blooms (CyanoHABs) originate from the excessive growth or bloom of cyanobacteria often referred to as blue-green algae. They have been on the rise globally in both marine and freshwaters in recently years with increasing frequency and severity owing to the rising temperature associated with climate change and increasing anthropogenic eutrophication from agricultural runoff and urbanization. Humans are at a great risk of exposure to toxins released from CyanoHABs through drinking water, food, and recreational activities, making CyanoHAB toxins a new class of contaminants of emerging concern. We investigated the toxic effects and mechanisms of microcystin-LR (MC-LR), the most prevalent CyanoHAB toxin, on the ovary and associated reproductive functions. Mouse models with either chronic daily oral or acute intraperitoneal exposure, an engineered three-dimensional ovarian follicle culture system, and human primary ovarian granulosa cells were tested with MC-LR of various dose levels. Single-follicle RNA sequencing, reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, western blotting, immunohistochemistry (IHC), and benchmark dose modeling were used to examine the effects of MC-LR on follicle maturation, hormone secretion, ovulation, and luteinization. Mice exposed long term to low-dose MC-LR did not exhibit any differences in the kinetics of folliculogenesis, but they had significantly fewer corpora lutea compared with control mice. Superovulation models further showed that mice exposed to MC-LR during the follicle maturation window had significantly fewer ovulated oocytes. IHC results revealed ovarian distribution of MC-LR, and mice exposed to MC-LR had significantly lower expression of key follicle maturation mediators. Mechanistically, in both murine and human granulosa cells exposed to MC-LR, there was reduced protein phosphatase 1 (PP1) activity, disrupted PP1-mediated PI3K/AKT/FOXO1 signaling, and less expression of follicle maturation-related genes. Using both invivo and in vitro murine and human model systems, we provide data suggesting that environmentally relevant exposure to the CyanoHAB toxin MC-LR interfered with gonadotropin-dependent follicle maturation and ovulation. We conclude that MC-LR may pose a nonnegligible risk to women's reproductive health by heightening the probability of irregular menstrual cycles and infertility related to ovulatory disorders. https://doi.org/10.1289/EHP12034.

Microcystin-LR, a Cyanobacterial Toxin, Induces Changes in the Organization of Membrane Compartments in Arabidopsis

To evaluate the effects of the cyanobacterial toxin microcystin-LR (MCY-LR, a protein phosphatase inhibitor) and diquat (DQ, an oxidative stress inducer) on the organization of tonoplast, the effect of MCY-LR on plastid stromule formation and on mitochondria was investigated in wild-type Arabidopsis. Tonoplast was also studied in PP2A catalytic (c3c4) and regulatory subunit mutants (fass-5 and fass-15). These novel studies were performed by CLSM microscopy. MCY-LR is produced during cyanobacterial blooms. The organization of tonoplast of PP2A mutants of Arabidopsis is much more sensitive to MCY-LR and DQ treatments than that of wild type. In c3c4, fass-5 and fass-15, control and treated plants showed increased vacuole fragmentation that was the strongest when the fass-5 mutant was treated with MCY-LR. It is assumed that both PP2A/C and B" subunits play an important role in normal formation and function of the tonoplast. In wild-type plants, MCY-LR affects mitochondria. Under the influence of MCY-LR, small, round-shaped mitochondria appeared, while long/fused mitochondria were typical in control plants. Presumably, MCY-LR either inhibits the fusion of mitochondria or induces fission. Consequently, PP2A also plays an important role in the fusion of mitochondria. MCY-LR also increased the frequency of stromules appearing on chloroplasts after 1 h treatments. Along the stromules, signals can be transported between plastids and endoplasmic reticulum. It is probable that they promote a faster response to stress.

Improved sensitivity of the anti-microcystin-LR ELISA using phage-displayed alpha-type anti-idiotypic nanobody

Anti-idiotypic antibodies (Ab2) are valuable tools that can be used for a better understanding of molecular mimicry and the immunological network. In this work, we showed a new application of a phage-displayed alpha-type Ab2 (Ab2α) to improve the sensitivity of an enzyme-linked immunosorbent assay (ELISA) detecting cyanobacterial toxin microcystin-LR (MC-LR). A monoclonal antibody (mAb) against MC-LR was used as an antigen to isolate binders in a camelid nanobody library. After three rounds of panning, three unique clones with strong binding against anti-MC-LR mAbs were isolated. These clones could specifically bind to anti-MC-LR mAbs without influencing mAbs binding with MC-LR, meaning these clones were Ab2αs. Based on the signal amplification effect of phage coat proteins and the non-competitive nature of Ab2α, a novel competitive ELISA method for MC-LR was established with a phage-displayed Ab2α. It showed that the phage-displayed Ab2α greatly enhanced the ELISA signal and sensitivity of the method was improved 3.5-fold to the conventional one. Combining with the optimization of pre-incubation time, the optimized ELISA decreased its limit of detection (LOD) from 4.5 ng/mL to 0.8 ng/mL (5.6-fold improvement). This new application of Ab2α may potentially be employed to improve the sensitivity of immunoassays for other environmental pollutants.

Searching for Optimal Substitute Habitats for Plants by Biological Experiments-A Case Study of the Endangered Species Aldrovanda vesiculosa L. (Droseraceae).

The selection of appropriate locations for the reintroduction of endangered plant species is an important process, because it usually influences the success of the conservation. The aim of this study was to select the optimal substitute habitats for Aldrovanda vesiculosa, taking into account the influence of physical–chemical factors (light intensity, temperature, pH, concentration of dissolved forms of nitrogen and cyanobacterial toxin microcystin-LR) on the efficiency of plant growth. Water analysis and field observations of the habitats of six lakes in Eastern Poland typified as potential substitute habitats for aldrovanda were carried out. The results of the experiments showed that both the concentration and the form in which nitrogen compounds are present in the environment were the factors limiting the growth rate and condition of plants. The second factor that caused the inhibition of aldrovanda growth was microcystin-LR. It was found that the habitat conditions in Lake Brzeziczno were within the ecological tolerance of the species. Particularly important was the low content of mineral compounds and the available forms of nitrogen and phosphorus in the water. Therefore, the probability of development of toxic cyanobacteria, the metabolites of which may affect the growth of A. vesiculosa, is also minimal.

Potentially Poisonous Plastic Particles: Microplastics as a Vector for Cyanobacterial Toxins Microcystin-LR and Microcystin-LF.

The potential of microplastics to act as a vector for micropollutants of natural or anthropogenic origin is of rising concern. Cyanobacterial toxins, including microcystins, are harmful to humans and wildlife. In this study, we demonstrate for the first time the potential of microplastics to act as vectors for two different microcystin analogues. A concentration of up to 28 times from water to plastic was observed for the combination of polystyrene and microcystin-LF achieving toxin concentrations on the plastic of 142 ± 7 μg g-1. Based on the experimental results, and assuming a worst-case scenario, potential toxin doses for daphnids are calculated based on published microplastic ingestion data. Progressing up through trophic levels, theoretically, the concentration of microcystins in organisms is discussed. The experimental results indicate that adsorption of microcystins onto microplastics is a multifactorial process, depending on the particle size, the variable amino acid composition of the microcystins, the type of plastic, and pH. Furthermore, the results of the current study stressed the limitations of exclusively investigating microcystin-LR (the most commonly studied microcystin congener) as a model compound representing a group of around 250 reported microcystin congeners.

Microcystin-LR, a Cyanobacterial Toxin, Induces DNA Strand Breaks Correlated with Changes in Specific Nuclease and Protease Activities in White Mustard (Sinapis alba) Seedlings.

There is increasing evidence for the induction of programmed cell death (PCD) in vascular plants by the cyanobacterial toxin microcystin-LR (MC-LR). Our aim was to detect the occurrence of PCD-related DNA strand breaks and their possible connections to specific nuclease and protease activities. DNA breaks were studied by the deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method in the photoperiodically grown dicot model of white mustard (Sinapis alba). In-gel nuclease and protease activity assays showed changes in the activities of specific isoenzymes during treatments with MC-LR. Strand breaks occurred both in the developing root epidermis and cortex. Several isoenzyme activities were related to these breaks, for example: an increase in the activity of neutral 80–75 kDa, acidic high MW (100–120 kDa) and, most importantly, an increase in the activity of neutral 26–20 kDa nucleases, all of them having single-stranded DNA cleaving (SSP nuclease) activities. Increases in the activities of alkaline proteases in the 61–41 kDa range were also detected and proved to be in relation with MC-LR-induced PCD. This is one of the first pieces of evidence on the correlation of PCD-related DNA strand breaks with specific hydrolase activities in a model dicot treated with a cyanobacterial toxin known to have environmental importance.

Cyanobacteria Microcystis aeruginosa Contributes to the Severity of Fish Diseases: A Study on Spring Viraemia of Carp.

Fish are exposed to numerous stressors in the environment including pollution, bacterial and viral agents, and toxic substances. Our study with common carps leveraged an integrated approach (i.e., histology, biochemical and hematological measurements, and analytical chemistry) to understand how cyanobacteria interfere with the impact of a model viral agent, Carp sprivivirus (SVCV), on fish. In addition to the specific effects of a single stressor (SVCV or cyanobacteria), the combination of both stressors worsens markers related to the immune system and liver health. Solely combined exposure resulted in the rise in the production of immunoglobulins, changes in glucose and cholesterol levels, and an elevated marker of impaired liver, alanine aminotransferase (ALT). Analytical determination of the cyanobacterial toxin microcystin-LR (MC-LR) and its structurally similar congener MC-RR and their conjugates showed that SVCV affects neither the levels of MC in the liver nor the detoxification capacity of the liver. MC-LR and MC-RR were depurated from liver mostly in the form of cysteine conjugates (MC-LR-Cys, MC-RR-Cys) in comparison to glutathione conjugates (LR-GSH, RR-GSH). Our study brought new evidence that cyanobacteria worsen the effect of viral agents. Such inclusion of multiple stressor concept helps us to understand how and to what extent the relevant environmental stressors co-influence the health of the fish population.

Whole-Genome Sequencing of Bacterial Isolates That Degrade the Cyanobacterial Toxin Microcystin-LR.

We previously demonstrated that 13 bacterial isolates from Lake Erie, when grown in groups of four to five isolates per group, degraded the cyanobacterial toxin microcystin-LR (MC-LR) into nontoxic fragments. Whole-genome sequencing of these bacteria was performed to provide genus and species information and to predict putative MC-LR-degrading genes.

Translocation of the cyanobacterial toxin microcystin-LR into guttation drops of Triticum aestivum and remaining toxicity

Uptake of the commonly occurring cyanobacterial toxin microcystin-LR (MC-LR) into crop plants via spray irrigation has been demonstrated. As other hazardous compounds such as pesticides were shown to be transported within plants, it was essential to understand the transport and fate of MC-LR in plants and the risks posed to grazers and other consumers. Of specific interest was to investigate if MC-LR could be detected in guttation drops and the toxicity thereof. Triticum aestivum (wheat) seedlings were exposed to 100 μg L−1 MC-LR in two separate experiments during which guttation drops were collected at various time points. The plants of one experiment were sectioned to investigate MC-LR distribution to the various plant appendages via liquid chromatography-tandem mass spectrometry analysis. After exposure, MC-LR could be detected in the roots, stems, leaves, and the guttation drops. However, the guttation drops were not toxic to Daphnia. As the environmentally relevant toxin concentration used was not sufficient to promote mortality in Daphnia, the physiological effect in insects, which rely on guttation drops as a water source, remains unknown. Combined with other contaminants that insects may be exposed to, the additional MC-LR exposure could contribute to the overall toxicity through the “tears of death”.

Effect of vacuum UV irradiation on the concentration of dissolved cyanobacterial toxin microcystin-LR

Abstract This study investigated the capability of vacuum UV to reduce the concentration of cyanobacterial toxin microcystin-LR (MC-LR) using low-pressure Hg lamps emitting 185 nm and 254 nm light. A collimated beam setup was used to irradiate samples of MC-LR solutions prepared in Milli-Q® water. The impact of competing water compounds was tested using solutions containing dissolved organic carbon (DOC), alkalinity (NaHCO3), and chloride (NaCl). Results showed that MC-LR in pure water at typical concentrations found in cyanobacterial bloom waters (17 and 40 μg/L) could be reduced below detection limits (0.5 μg/L) within one minute of irradiation time by a UV dose less than 40 mJ/cm2. A solution with a much higher initial concentration of MC-LR (870 μg/L) did show a reduced degradation rate. The presence of competing compounds does appear to reduce observed MC-LR degradation rates with the greatest impact caused by DOC followed by alkalinity followed by chloride. MC-LR degradation appears to occur by both direct photolysis by 254 nm photons and by advanced oxidation by hydroxyl radicals generated from 185 nm photons. Vacuum UV has shown promising capability at reducing MC-LR concentration.

Potential role of engineered nanoparticles as contaminant carriers in aquatic ecosystems: Estimating sorption processes of the cyanobacterial toxin microcystin-LR by TiO2 nanoparticles

Abstract In the present study, the adsorption of a cyanobacterial toxin, microcystin-LR (MC-LR), onto titanium dioxide nanoparticles (TiO 2 -NPs) was investigated in aqueous solution. TiO 2 -NPs with different crystalline phases (anatase, rutile, anatase-rutile mixture) and MC-LR in an environmental relevant concentration for German water bodies were used to make results more applicable to environmental conditions. To investigate the adsorption mechanism as well as to determine the rate-limiting step of adsorption, analysis of the adsorption kinetic was performed and common kinetic models were applied to the experimental data. Additionally, the influence of nano-TiO 2 dosage (0.01–10 mg/L) and particle size on adsorption capacity was examined, the latter by comparing the adsorption data of TiO 2 -NPs to a bulk sized counterpart. The analysis of the adsorption kinetic indicates a complex adsorption mechanism and the involvement of pseudo-second-order chemisorption in the adsorption process. While only anatase TiO 2 showed a variation of MC-LR adsorption at different adsorbent concentrations, the particle size of the adsorbents appears to be the most influencing factor on toxin adsorption. All TiO 2 -NPs exhibited higher adsorption capacities compared to the bulk TiO 2 counterpart, which was statistically significant for the environmental relevant concentration levels of TiO 2 -NPs. The obtained results emphasise the potential of TiO 2 -NPs to act as carriers for environmental contaminants such as the cyanobacterial toxin MC-LR, and thus to influence its fate in aquatic ecoystems.

Single and combined exposure to MC-LR and BMAA confirm suitability of Aegagropila linnaei for use in green liver systems®–A case study with cyanobacterial toxins

The filamentous green algae Aegagropila linnaei was tested for its uptake capacity of the cyanobacterial toxins microcystin-LR (MC-LR) and β-N-methylamino-l-alanine (BMAA) in order to approve the suitability of its use in the Green Liver System®. Uptake into the algae and toxin reduction in the medium were analyzed by LC–MS/MS after static exposure for one week to 20μgL−1 MC-LR, 80μgL−1 BMAA, and 20μgL−1 MC-LR together with 80μgL−1 BMAA, respectively. BMAA was effectively removed by A. linnaei within 5 days compared to only around 35% removal of the initial exposure concentration in the case of MC-LR, independent of the application mode, in single or in a mixture. However, differences were found for BMAA amounts taken up into the tissue in that it was higher if applied in combination with MC-LR.Additionally, physiological responses such as the activity of biotransformation enzyme glutathione S-transferase (GST), antioxidant enzymes peroxidase (POD) and catalase (CAT) as well as the development of the reactive oxygen species hydrogen peroxide (H2O2) were compared between the different treatment groups in order to determine possible harmful effects of the toxin exposure on the algae. In contrast to the toxin exposure to a single toxin with no significant enzymatic response, exposure to the toxin mixture provoked an immediate increase in GST and CAT activity after one day as well as after longer exposure for one week, hinting on an enhanced need for prevention against exposure derived reactive oxygen species as well as putative biotransformation attempts in a mixture exposure scenario.

Response surface modeling and optimization of microcystin-LR removal from aqueous phase by polyacrylamide/sodium alginate–montmorillonite superabsorbent nanocomposite

The adsorptive removal of the most commonly occurring cyanobacterial toxin microcystin–LR by polyacrylamide/sodium alginate-modified montmorillonite (PAM/SA-MMT) superabsorbent nanocomposite was investigated through the HPLC/UV system. The process of PAM/SA-MMT preparation and MC-LR adsorption was characterized by scanning electron microscopy, energy dispersive X-ray microanalysis, X-ray diffraction analysis, and Fourier transform infrared spectroscopy analysis. With the application of Box–Behnken statistical experimental design combined with Response surface methodology, the quadratic statistical model was established to predict the interactive effects of pH, weight ratio (wr) of AM to SA, and weight content (wt) of MMT on MC-LR adsorption and to optimize the main controlling parameters. The maximum adsorption capacity was observed with pH 2.5–4.5, wr 55–65, and wt3–5%. The MC-LR adsorption capacity of PAM/SA-MMT increased with increase in temperature from 10 to 40°C. Kinetics were consistent with Pseudo models and revealed that the sorption process could reach the equilibrium within 80 min and involved several different rate-controlling kinetic stages. The Langmuir isotherm model predicted that the theoretical maximum adsorption capacity of PAM/SA-MMT was 32.66 mg g−1. Over 85% adsorption and 80% desorption could be achieved after five regeneration cycles and the recovery of MC-LR reached 92.8% without ionic effect. PAM/SA-MMT superabsorbent nanocomposite was determined as effective, economic, and environmentally benign for MC-LR removal on a large-scale application.

Evaluation of the photocatalytic activity of TiO2 based catalysts for the degradation and mineralization of cyanobacterial toxins and water off-odor compounds under UV-A, solar and visible light

Research on the development of new TiO2 based photocatalysts has been receiving increased attention due to the ability of TiO2 to degrade a great variety of organic compounds upon UV-A irradiation. In order to evaluate the photocatalytic performance of the new synthesized materials, it is essential to follow specific procedures during the photocatalytic process. Special care should be given on light intensity, presence of oxygen, catalyst loading, initial concentration of substrate, adsorption, pH, different irradiation wavelength, mineralization, intermediate products and toxicity. In this study, catalysts such as commercially available materials (Degussa P25, Kronos vlp-7000) and home prepared materials (N-TiO2, GO–TiO2 and Ref-TiO2) have been tested for their photocatalytic ability on the degradation and mineralization of the cyanobacterial toxin microcystin-LR and off-odor causing compounds (geosmin, 2-methylisoborneol) under UV-A, solar and visible light irradiation. Also, the identification of intermediate products and their toxicity under different experimental conditions for microcystin-LR was studied. Our results showed that in all cases of the compounds Degussa P25 was the better performing catalyst under UV-A light irradiation. Under solar light, all compounds were effectively degraded with the doped materials (N-TiO2, GO–TiO2, Kronos vlp-7000) showing better photocatalytic performance than theirs undoped material (Ref-TiO2). As far as concerning visible light irradiation, only the visible light activated materials showed some photocatalytic activity (N-TiO2, Kronos vlp-7000). It was also showed in order to have reproducible evaluation results on the photocatalytic performance of several catalysts (intra and inter-laboratory), a careful selection of experimental parameters is required.

Proton and Iron Binding by the Cyanobacterial Toxin Microcystin-LR

Microcystins (MCs) are a group of hepatotoxins produced by cyanobacteria that have not had their functional role or the environmental factors that trigger production clearly determined. One suggestion is that microcystins are siderophores (i.e., ligands with an extremely high affinity with iron, typically with stability constants substantially greater than 10(25)). In this work, we explore proton and iron binding with microcystin-LR (MC-LR). Using UV-visible spectroscopy and a HPLC peak retention time-based method, the two acid dissociation constants associated with the carboxylic groups of MC-LR were determined to be: pKa₁ = 2.17 and pKa₂ = 3.96. Cyclic voltammetry provides evidence for the formation of at least two Fe(III)-MC-LR complexes, with the Fe(III) reduction peak significantly shifted to more reducing potentials in the presence of MC-LR. These complexes have been interpreted as a rapidly formed initial complex (Complex 1) and a more stable, and slower forming, Complex 2. The stability constant for Fe(III)-MC-LR (Complex 2) was estimated to be approximately 10(13) in 60% v/v MeOH/water at 0.1 M ionic strength. The electrochemical experiments provide no evidence for the formation of a complex between Fe(2+) and MC-LR. Given that most MC-LR is released only upon cell lysis, and coupled with the moderate strength of the stability constant with Fe(III) determined in this study, it appears unlikely that that MC-LR is an extracellular siderophore. If MC-LR is involved in iron regulation in cyanobacteria, it is more likely as a shuttle for iron across the cell membrane or in intracellular processes.

Histological, cytological and biochemical alterations induced by microcystin-LR and cylindrospermopsin in white mustard (Sinapis albaL.) seedlings

This study compares the histological, cytological and biochemical effects of the cyanobacterial toxins microcystin-LR (MCY-LR) and cylindrospermopsin (CYN) in white mustard (Sinapis alba L.) seedlings, with special regard to the developing root system. Cyanotoxins induced different alterations, indicating their different specific biochemical activities. MCY-LR stimulated mitosis of root tip meristematic cells at lower concentrations (1 μg ml-1) and inhibited it at higher concentrations, while CYN had only inhibitory effects. Low CYN concentrations (0.01 μg ml-1) stimulated lateral root formation, whereas low MCY-LR concentrations increased only the number of lateral root primordia. Both inhibited lateral root development at higher concentrations. They induced lignifications, abnormal cell swelling and inhibited xylem differentiation in roots and shoots. MCY-LR and CYN induced the disruption of metaphase and anaphase spindles, causing altered cell divisions. Similar alterations could be related to decreased protein phosphatase (PP1 and PP2A) activities in shoots and roots. However, in vitro phosphatase assay with purified PP1 catalytic subunit proved that CYN in contrast to MCY-LR, decreased phosphatase activities of mustard in a non-specific way. This study intends to contribute to the understanding of the mechanisms of toxic effects of a protein phosphatase (MCY-LR) and a protein synthesis (CYN) inhibitory cyanotoxin in vascular plants.

Determination of microcystin-LR in water by a label-free aptamer based electrochemical impedance biosensor

In this study, an electrochemical impedance biosensor for cyanobacterial toxin microcystin-LR (MC-LR) detection has been developed. MC-LR aptamers were immobilized on the gold electrode through Au–S interaction, in the presence of target (MC-LR); the binding of MC-LR and aptamers probe led to a complex formation change on the electrode surface and resulted in the impedance decreasing. The decrease rate had a linear relationship with logarithm of the MC-LR concentration in the range of 1.0×10−7–5.0×10−11mol/L, with a detection limit of 1.8×10−11mol/L. The sensor has good selectivity and stability, it has been applied to detect MC-LR in three kinds of real water samples with satisfying results.

Multiplex competitive microbead-based flow cytometric immunoassay using quantum dot fluorescent labels

In answer to the ever-increasing need to perform the simultaneous analysis of environmental hazards, microcarrier-based multiplex technologies show great promise. Further integration with biofunctionalized quantum dots (QDs) creates new opportunities to extend the capabilities of multicolor flow cytometry with their unique fluorescence properties. Here, we have developed a competitive microbead-based flow cytometric immunoassay using QDs fluorescent labels for simultaneous detection of two analytes, bringing the benefits of sensitive, rapid and easy-of-manipulation analytical tool for environmental contaminants. As model target compounds, the cyanobacterial toxin microcystin-LR and the polycyclic aromatic hydrocarbon compound benzo[a]pyrene were selected. The assay was carried out in two steps: the competitive immunological reaction of multiple targets using their exclusive sensing elements of QD/antibody detection probes and antigen-coated microsphere, and the subsequent flow cytometric analysis. The fluorescence of the QD-encoded microsphere was thus found to be inversely proportional to target analyte concentration. Under optimized conditions, the proposed assay performed well within 30min for the identification and quantitative analysis of the two environmental contaminants. For microcystin-LR and benzo[a]pyrene, dose–response curves with IC50 values of 5μgL−1 and 1.1μgL−1 and dynamic ranges of 0.52–30μgL−1 and 0.13–10μgL−1 were obtained, respectively. Recovery was 92.6–106.5% for 5 types of water samples like bottled water, tap water, surface water and seawater using only filtration as sample pretreatment.

Integrated Histopathological and Urinary Metabonomic Investigation of the Pathogenesis of Microcystin-LR Toxicosis

Patterns of change of endogenous metabolites may closely reflect systemic and organ-specific toxic changes. The authors examined the metabolic effects of the cyanobacterial (blue-green algal) toxin microcystin-LR by (1)H-nuclear magnetic resonance (NMR) analysis of urinary endogenous metabolites. Rats were treated with a single sublethal dose, either 20 or 80 µg/kg intraperitoneally, and sacrificed at 2 or 7 days post dosing. Changes in the high-dose, 2-day sacrifice group included centrilobular hepatic necrosis and congestion, accompanied in some animals by regeneration and neovascularization. By 7 days, animals had recovered, the necrotizing process had ended, and the centrilobular areas had been replaced by regenerative, usually hypertrophic hepatocytes. There was considerable interanimal variation in the histologic process and severity, which correlated with the changes in patterns of endogenous metabolites in the urine, thus providing additional validation of the biomarker and biochemical changes. Similarity of the shape of the metabolic trajectories suggests that the mechanisms of toxic effects and recovery are similar among the individual animals, albeit that the magnitude and timing are different for the individual animals. Initial decreases in urinary citrate, 2-oxoglutarate, succinate, and hippurate concentrations were accompanied by a temporary increase in betaine and taurine, then creatine from 24 to 48 hours. Further changes were an increase in guanidinoacetate, dimethylglycine, urocanic acid, and bile acids. As a tool, urine can be repeatedly and noninvasively sampled and metabonomics utilized to study the onset and recovery after toxicity, thus identifying time points of maximal effect. This can help to employ histopathological examination in a guided and effective fashion.

A Membrane-Based ELISA Assay and Electrochemical Immunosensor for Microcystin-LR in Water Samples

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.