Presence Of microcystin-LR Research Articles

Biodegradation studies and sequencing of microcystin-LR degrading bacteria isolated from a drinking water biofilter and a fresh water lake

The presence of microcystin-LR -degrading bacteria in an active anthracite biofilter and in Lake Mead, Nevada was investigated. Four bacterial isolates from enrichment culture were identified using 16S rRNA analysis. Microcystin biodegradation tests were performed with both, the enrichment cultures and the respective isolates, using microcystin alone and acetate as carbon sources. A newly recognized microcystin-degrading bacterium, Morganella morganii, was isolated from the biofilter and from Lake Mead. The results of the biodegradation tests indicated that addition of a carbon source (acetate), significantly repressed the degradation of microcystin-LR. The findings of this study inform on the prevalence of microcystin-degrading bacteria in the environment indicating bioaugmentation may not be needed, if biofiltration is used to remove microcystin from waters. The results also imply that, in a biofilter, biodegradable naturally organic matter (NOM) and microcystin will compete and therefore lower toxin removals are likely in waters with higher NOM content. The feasibility of removing microcystin by biofiltration depends on the toxin concentration and the concentration of biodegradable carbon sources in the biofilter.

The influence of microcystin-LR on fish phagocytic cells

The in vitro influence of microcystin-LR (MC-LR) on rainbow trout phagocytic cell functions was studied. The cells isolated from the fish blood were exposed on MC-LR at the concentrations of 1, 5, 10, 20 microg/mL. The viability of the cells after 2, 4 and 24 hour incubation with MC-LR was studied as well as phagocytic cell ability measured as zymozan particle phagocytosis and metabolic activity measured as respiratory burst activity. The toxin caused time- and dose-dependent viability decrease, the strongest after cell exposure on 10 and 20 microg/mL and observed only after 24 hours incubation when 1 microg/mL was applied. The phagocytic ability was elevated in the presence of MC-LR at the dose of 5 microg/mL. MC-LR had also modulatory influence on respiratory burst activity. While the two lower doses caused the stimulation of the parameter, the two higher ones diminished superoxide anion production in the triggered cells. The results show the potency of the toxin to influence the fish phagocytic cell functions.

A fluorescent-labeled microcystin-LR terbium cryptate

A new fluorescent labeled compound of microcystin-LR with terbium cryptate was obtained by initial conjugation of microcystins-LR with aminoethanethiol followed by the reaction with the ester group of terbium cryptate. The product formation was followed by high performance liquid chromatography (HPLC) at 238 nm and 310 nm. The presence of microcystin-LR in the labeled molecule was confirmed by enzyme linked immunosorbent assay ELISA and by protein reaction with bicinhonic acid. Luminescence spectra of cryptate and the conjugated molecule were carried through as well.

Effects of microcystins on and toxin degradation by Poterioochromonas sp.

A Chrysophyceae species, Poterioochromonas sp., was isolated from Microcystis cultures. This species can efficiently prey on Microcystis and can grow faster phagotrophically than autotrophically. The growth of Poterioochromonas sp. was stimulated in the presence of microcystin-LR and microcystin-RR (in concentrations ranging from 0.1 to 4 mg/L). The growth rate of Poterioochromonas was 4-5 times higher than the control, indicating the toxins serve as growth stimuli for this organism. A subculture of toxin-treated cells, however, showed low cellular viability, suggesting that growth enhancement by microcystins was not a normal process. The antioxidant enzymatic activity of Poterioochromonas sp. was screened for toxicology analysis. Glutathione, malondialdehyde, and superoxide dismutase (SOD) content was up-regulated within 8 h of exposure to microcystin-LR (500 microg/L). A high level of SOD activity during exposure to the toxin indicated that SOD was involved in decreasing oxidative stress caused by microcystin-LR. Simultaneously with growth, Poterioochromonas was able to degrade microcystin-LR even, at a toxin concentration of 4 mg/L. This putative degradation mechanism in Poterioochromonas is explored further and discussed in this article. Our findings may shed light on understanding the role of Poterioochromonas in the aquatic ecosystem, in particular, as a grazer of toxic cyanobacteria and a biodegrader for microcystins.

First observation of microcystin-LR in pelagic cyanobacterial blooms in the northern Baltic Sea

Cyanobacterial bloom samples from the Gulf of Finland (northern Baltic Sea) were collected in July 2003 and analyzed for microcystins and nodularins, cyanobacterial peptide hepatotoxins, by ELISA, HPLC-UV and LC-MS. The blooms consisted mainly of the genera Nodularia, Anabaena and Aphanizomenon. The main hepatotoxin in the samples was nodularin-R (Nod-R), all the samples also contained demethylnodularin-R. The presence of microcystin-LR was confirmed in three locations out of nine by multiple reactant monitoring on the triple quadrupole mass spectrometer. This is the first reported finding of microcystins in the Baltic Sea from the open sea area. Anabaena was the likely producer of microcystin-LR in the samples.

The effect of intraperitoneally administered microcystin-LR on the gastrointestinal tract of Balb/c mice.

Microcystin-LR (MCLR) has been associated with the development of gastrointestinal complaints in people ingesting cyanobacterial bloom contaminated water. The symptoms usually present a day or two after exposure raising questions as to the toxic effects of MCLR on the gastrointestinal tract. This study investigated the apoptotic effect of ip administered MCLR over time on the duodenum, jejenum and ileum of mice receiving a single 75% LD 50 dose. The apoptotic index was significantly raised in all sections at 8 h post exposure (pe) and continued to rise for the 16, 24 and 32 h pe groups, while the glycogen levels were normal at 24 h pe. The duodenum exhibited the most significant increase in apoptotic index overall, followed by the jejenum and ileum. Immunohistochemistry indicated the presence of MCLR in the lamina propria suggesting a role for MCLR in the induction of apoptosis in the GIT of mice exposed to a single sublethal dose of MCLR.

Microcystin-LR affects cytoskeleton and morphology of rabbit primary whole embryo cultured cells in vitro

Microcystin-LR is the most frequently studied cyclic heptapeptide produced by different genera of cyanobacteria and is hepatotoxic to livestock and human populations. The adverse effects of microcystin-LR on morphology and cytoskeletal elements in different stages of early embryonal development have been studied in vitro. Embryos and whole embryo cultures have been exposed to microcystin-LR (10–100 μM). Actin filaments were visualized by fluorescence staining and the microtubular network labelled by immunostaining. Growth, development and cytoskeleton organization of the embryos embedded in zona pellucida are not affected by microcystin-LR in concentrations up to 100 μM, while whole embryo cell cultures are affected by the presence of microcystin-LR in the culture medium. High microcystin-LR concentrations (100 μM) cause cells to be detached and destroyed, while lower concentrations (10–20 μM) profoundly affect actin and microtubule organization. These effects are confirmed also by the presence of transformed microcystin-LR in all the media at the lowest concentrations. It seems that the changes to the cells are far more serious than that expressed in cell morphology. From our experiments we conclude that the presence of zona pellucida is an effective way of embryo protection against xenobiotics like microcystin-LR.

Investigations into the inhibitory effects of microcystins on plant growth, and the toxicity of plant tissues following exposure

The cyanobacterial toxins microcystins are known to affect a number of processes in plant tissues, and their presence in water used for irrigation may have considerable impact on the growth and development of crop plants. In this study, two plant bioassays were employed to investigate the phytotoxic effects of microcystins. A plant tissue culture assay revealed that the growth and chlorophyll content of Solanum tuberosum L. cultures was inhibited at microcystin-LR concentrations of 0.005 and 0.05 μg cm −3 , respectively. A previously developed bioassay was also employed to determine the effects of three commonly occurring microcystin variants on the growth of Synapis alba L. seedlings. Microcystins-LR, -RR, and -LF inhibited the growth of seedlings, with GI 50 values of 1.9, 1.6 and 7.7 μg ml −1, respectively. The growth of Phaseolus vulgaris was also examined in the presence of microcystin-LR. The toxin was found to have little effect on growth for up to 18 days, but impaired the development of the roots of exposed plants, causing them to take up approximately 30% less growth medium than those grown in the absence of toxin. Microcystin was also detected in the tissues of exposed plants using a commercially available ELISA kit, suggesting that the uptake of these toxins by edible plants may have significant implications for human health.

The rise and fall of a toxigenic population of Microcystis aeruginosa (Cyanophyceae/Cyanobacteria)—a decade of observations in Lake Akersvatnet, Norway

Abstract The phytoplankton community of Lake Akersvatnet before 1968 was dominated by Planktothrix mougeotii (Bory ex Gomont) Anagnostidis et Komarek (= Oscillatoria agardhii Gomont var. isothrix Skuja). Non-filamentous species of cyanophytes, however, became subsequently frequent. Populations of Microcystis aeruginosa (Kutzing) Lemmermann exhibited regular mass development during the period 1968–1989. In summer 1984 the presence of microcystin-LR was detected during a nuisance bloom. Toxic blooms of M. aeruginosa were a recurrent phenomenon until 1989. The population of Microcystis was then suppressed, and microcystin was present in trace concentrations only or could not be detected. The phytoplankton was subsequently dominated by species of Anabaena and Aphanizomenon and in 1994 by the dinoflagellateCeratium hirundinella (O.F. Muller) Dujardin, Our results suggest an interrelation between changes in cyanophyte bloom composition and toxin production during various stages of eutrophication.

Regulation of flagellar dynein by an axonemal type-1 phosphatase in Chlamydomonas.

Physiological studies have demonstrated that flagellar radial spokes regulate inner arm dynein activity in Chlamydomonas and that an axonemal cAMP-dependent kinase inhibits dynein activity in radial spoke defective axonemes. These studies also suggested that an axonemal protein phosphatase is required for activation of flagellar dynein. We tested whether inhibitors of protein phosphatases would prevent activation of dynein by the kinase inhibitor PKI in Chlamydomonas axonemes lacking radial spokes. As predicted, preincubation of spoke defective axonemes (pf14 and pf17) with ATP gamma S maintained the slow dynein-driven microtubule sliding characteristic of paralyzed axonemes lacking spokes, and blocked activation of dynein-driven microtubule sliding by subsequent addition of PKI. Preincubation of spoke defective axonemes with the phosphatase inhibitors okadaic acid, microcystin-LR or inhibitor-2 also potently blocked PKI-induced activation of microtubule sliding velocity: the non-inhibitory okadaic acid analog, 1-norokadaone, did not. ATP gamma S or the phosphatase inhibitors blocked activation of dynein in a double mutant lacking the radial spokes and the outer dynein arms (pf14pf28). We concluded that the axoneme contains a type-1 phosphatase required for activation of inner arm dynein. We postulated that the radial spokes regulate dynein through the activity of the type-1 protein phosphatase. To test this, we performed in vitro reconstitution experiments using inner arm dynein from the double mutant pf14pf28 and dynein-depleted axonemes containing wild-type radial spokes (pf28). As described previously, microtubule sliding velocity was increased from approximately 2 microns/second to approximately 7 microns/second when inner arm dynein from pf14pf28 axonemes ws reconstituted with axonemes containing wild-type spokes. In contrast, pretreatment of inner arm dynein from pf14pf28 axonemes with ATP gamma S, or reconstitution in the presence of microcystin-LR, blocked increased velocity following reconstitution, despite the presence of wild-type radial spokes. We conclude that the radial spokes, through the activity of an axonemal type-1 phosphatase, activate inner arm dynein by dephosphorylation of a critical dynein component. Wild-type radial spokes also operate to inhibit the axonemal cAMP-dependent kinase, which would otherwise inhibit axonemal dynein and motility.

Inhibition of protein phosphatases activates glucose-6-phosphatase in isolated rat hepatocytes

Incubation of hepatocytes in the presence of microcystin-LR, okadaic acid, calyculin A (inhibitors of protein phosphatases PP1 and PP2A) or microcystin-RR (a specific inhibitor of PP2A) activated glucose-6-phosphatase both in the supernatant and in intact or disrupted microsomes. Puromycin, an inhibitor of protein synthesis, totally suppressed this activating effect, suggesting the involvement of protein phosphatases in the regulation of glucose-6-phosphatase synthesis.