Kenneth A. Beattie

Cyanobacterial microcystin‐LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants

The cyclic heptapeptide, microcystin‐LR, inhibits protein phosphatases 1 (PP1) and 2A (PP2A) with K i, values below 0.1 nM. Protein phosphatase 2B is inhibited 1000‐fold less potently, while six other phosphatases and eight protein kinases tested are unaffected. These results are strikingly similar to those obtained with the tumour promoter okadaic acid. We establish that okadaic acid prevents the binding of microcystin‐LR to PP2A, and that protein inhibitors 1 and 2 prevent the binding of microcystin‐LR to PP1. We discuss the possibility that inhibition of PP1 and PP2A accounts for the extreme toxicity of microcystin‐LR, and indicate its potential value in the detection and analysis of protein kinases and phosphatases.

Identification of an enzymatically formed glutathione conjugate of the cyanobacterial hepatotoxin microcystin-LR: the first step of detoxication

Cyanobacterial toxins have adverse effects on mammals, birds and fish and are being increasingly recognised as a potent stress factor and health hazard factor in aquatic ecosystems. Microcystins, cyclic heptapeptides and a main group of the cyanotoxins are mainly retained within the producer cells during cyanobacterial bloom development. However, these toxins are released into the surrounding medium by senescence and lysis of the blooms. Any toxin present could then come into contact with a wide range of aquatic organisms including phytoplankton grazers, invertebrates, fish and aquatic plants. Recent studies showed the conversion of microcystin in animal liver to a more polar compound in correlation with a depletion of the glutathione pool of the cell. The present study shows the existence of a microcystin-LR glutathione conjugate formed enzymatically via soluble glutathione S-transferase in various aquatic organisms ranging from plants (Ceratophyllum demersum), invertebrates (Dreissena polymorpha, Daphnia magna) up to fish eggs and fish (Danio rerio). The main derived conjugate was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry yielding a mass of m/z 1302, which is equivalent to the mass assumed for a glutathione microcystin-LR conjugate. This conjugate appears to be the first step in the detoxication of a cyanobacterial toxin in aquatic organisms.

Cyanobacterial toxins, exposure routes and human health

The production of potent toxins by bloom-, scum- and mat-forming cyanobacteria, in fresh-, brackish and marine waters, appears to be a global phenomenon. Cyanobacterial toxins can also be produced by cyanobacteria from terrestrial sources. The range and number of known cyanobacterial toxins are increasing apace as associated poisoning incidents are investigated, and increasingly powerful analytical methods are applied to complement toxicity-based studies on both natural samples and laboratory isolates of cyanobacteria. Water quality management to reduce toxic cyanobacterial mass developments, and schemes to mitigate the potential effects of cyanobacterial toxins, require an understanding of the occurrence and properties of the toxins and of the exposure routes via which the toxins present risks to health. Here, we review advances in the recognition of cyanobacterial toxins and their toxicity, and of the exposure routes with reference to human health, namely via skin contact, inhalation, haemodialysis and ...

Identification of anatoxin-A in benthic cyanobacteria (blue-green algae) and in associated dog poisonings at Loch Insh, Scotland

Dog deaths occurred in 1990 and 1991 after the animals drank water containing blooms of benthic cyanobacteria along the shoreline of Loch Insh, Scotland. Signs of poisoning in the affected animals and the high neurotoxicity of bloom extracts in laboratory bioassays indicated acute poisoning due to cyanobacterial neurotoxin(s). The neurotoxic blooms consisted largely of benthic Oscillatoria species which were also observed in the stomach contents of the poisoned dogs. Stomach contents were also neurotoxic in bioassays with the same signs of poisoning as the Oscillatoria blooms. The cyanobacterial alkaloid neurotoxin anatoxin-a was identified in bloom extracts and poisoned dog stomach contents by high-performance liquid chromatography and gas chromatography-mass spectrometry. A species of benthic Oscillatoria has been isolated from the neurotoxic bloom material and shown to produce anatoxin-a in laboratory culture. These findings are the first to associate anatoxin-a toxicoses with benthic, rather than planktonic, cyanobacteria. Procedures for anatoxin-a extraction and identification from the blooms and animal material are also detailed.

Keap1 perceives stress via three sensors for the endogenous signaling molecules nitric oxide, zinc, and alkenals.

Recognition and repair of cellular damage is crucial if organisms are to survive harmful environmental conditions. In mammals, the Keap1 protein orchestrates this response, but how it perceives adverse circumstances is not fully understood. Herein, we implicate NO, Zn2+, and alkenals, endogenously occurring chemicals whose concentrations increase during stress, in this process. By combining molecular modeling with phylogenetic, chemical, and functional analyses, we show that Keap1 directly recognizes NO, Zn2+, and alkenals through three distinct sensors. The C288 alkenal sensor is of ancient origin, having evolved in a common ancestor of bilaterans. The Zn2+ sensor minimally comprises H225, C226, and C613. The most recent sensor, the NO sensor, emerged coincident with an expansion of the NOS gene family in vertebrates. It comprises a cluster of basic amino acids (H129, K131, R135, K150, and H154) that facilitate S-nitrosation of C151. Taken together, our data suggest that Keap1 is a specialized sensor that quantifies stress by monitoring the intracellular concentrations of NO, Zn2+, and alkenals, which collectively serve as second messengers that may signify danger and/or damage.

Uptake and effects of microcystin-LR on detoxication enzymes of early life stages of the zebra fish (Danio rerio)

The effects of cyanotoxins on fish have been studied mainly in adults, rather than in early life stages which could be more sensitive or, in view of their immobility, more readily affected. The uptake of microcystin‐LR by different early life stages of the zebra fish (Danio rerio) was investigated using 14C‐labelled microcystin‐LR. The effects on the activity of the detoxication enzymes, microsomal and soluble glutathione S‐transferases (GST), and glutathione peroxidase (GP‐X) were examined. There was a detectable uptake of microcystin from the first day of embryonic development up to 5 day old larvae. On average, an absorption of 0.5 ng microcystin for eggs and eleuthero‐embryos was calculated over the entire exposure time. Because of the differences in volume of the eggs and eleuthero‐embryos, there was an increase in the microcystin‐LR concentration between these stages. In the eggs, approximately 25% of the medium concentration was found, and in eleuthero‐embryos an equilibrium between fish and medium was reached.

Identification of a microcystin in benthic cyanobacteria linked to cattle deaths on alpine pastures in Switzerland

During the last two decades, more than 100 cattle deaths have been reported from 11 alpine sites in south-eastern Switzerland. Pathological findings and the histological examination of their organs strongly indicated acute hepatotoxicosis. Clinical symptoms suggestive of neurotoxicity were also observed in some cases. To elucidate the etiology of these poisonings, different water bodies in one of the affected alpine pastures were investigated for cyanobacterial toxins. The waters were highly oligotrophic, cold and turbid, and the ice-free period was limited to 3–4 months. The algal community in these waters consisted mostly of benthic cyanobacteria forming dense mats on the surface of sediments and on submerged rocks. Oscillatoria limosa and Phormidium konstantinosum (=Oscillatoria tenuis) dominated these populations, but occasionally other species of Oscillatoria, Phormidium, Tychonema and Pseudanabaena also occurred in the mats. Samples from the cyanobacterial mats yielded positive results in a protein ...

Retention of Microcystis aeruginosa and microcystin by salad lettuce (Lactuca sativa) after spray irrigation with water containing cyanobacteria

Colonies and single cells of Microcystis aeruginosa and the hepatotoxin microcystin were retained by salad lettuce after growth with spray irrigation water containing the microcystin-producing cyanobacteria. These findings are discussed in terms of crop spray irrigation with water containing cyanobacteria and potential human exposure to cyanobacterial toxins via plant foods grown in such circumstances.

Comparative effects and metabolism of two microcystins and nodularin in the brine shrimp Artemia salina.

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.

Toxicity of cylindrospermopsin to the brine shrimp Artemia salina: comparisons with protein synthesis inhibitors and microcystins

The Artemia salina bioassay was successfully applied to the analysis of the hepatotoxic cyanobacterial alkaloid and protein synthesis inhibitor, cylindrospermopsin. A dose-dependent response in mortality was observed for purified cylindrospermopsin and LC(50) values decreased with time from 8.1 to 0.71 microg/ml(-1), between 24 and 72 h, respectively. Cylindrospermopsin was slightly less potent than micro cystin-LR, with similar LC(50) values on a gravimetric basis, but was more toxic to A.salina than the protein synthesis inhibitors, cycloheximide, chloramphenicol and tetracycline. Cylindrospermopsin-containing strains of the cyanobacterium Cylindrospermopsis raciborskii were found to be toxic to A.salina and the LC(50) concentration for these strains over time was greater than the LC(50) for purified cylindrospermopsin, with the exception of C. raciborskii strain CR1.