Attack of Microcystis aeruginosa bloom on a Ceratophyllum submersum field: Ecotoxicological measurements in real environment with real microcystin exposure.

Overproduction of toxic cyanobacteria is a type of harmful algal blooms (HABs). The heptapeptide microcystins (MCs) are one of the most common cyanotoxins. There is increasing research concerning the effects of MCs on growth and physiology of vascular plants, however there is a lack of studies on their direct effects on aquatic macrophytes in the real environment. Here we report the occurrence of a MC producing HAB in Lake Bárdos, Hungary in 2012 with harmful effects on cytological, histological and biochemical parameters of Ceratophyllum submersum (soft hornwort) plants naturally growing at the blooming site. Blue-Green Sinapis Test (BGST) showed high toxicity of HAB samples. Cell-free water samples contained a significant amount of MCs (7.31 ±â€¯0.17 µg L-1) while C. submersum plants contained 1.01 ±â€¯0.21 µg g DW-1 MCs. Plants showed significant increases of protein content and decreases of anthocyanin content and carotenoid/chlorophyll ratio, indicating physiological stress- as compared to plants from the control (MC free) sampling site of the same water body. Histological and cytological studies showed (i) radial swelling and the abnormal formation of lateral buds at the shoot tip leading to abnormal development; (ii) the fragmentation of nuclei as well as accumulation of phenolics in the nucleus indicating that the HAB induced cell death and stress reactions at the nuclear level. The most relevant effect was the increase of histone H3 phosphorylation in metaphase chromosomes: since MCs are strong inhibitors of protein phosphatases, this alteration is related to the biochemical targets of these toxins. The HAB decreased peroxidase activity, but increased nuclease and protease activities, showing the decreased capacity of plants to face biotic stress and as the cytological changes, the induction of cell death. This study is one of the first to show the complex harmful changes in aquatic plants that co-exist with HABs.

The possible role of microcystin (D-Leu1 MC-LR) as an antioxidant on Microcystis aeruginosa (Cyanophyceae). In vitro and in vivo evidence.

Microcystins constitute a serious threat to the quality of drinking water worldwide. However, the eco-physiological role of them is not completely known and it is suggested that toxins can play a role in the antioxidant protection. The objective of this study was to evaluate the microcystin antioxidant capacity in vitro by Electronic Paramagnetic Resonance, highly specific for the different reactive oxygen species and in vivo by 7 days exposure of Microcystis aeruginosa to high (29 °C) temperature in addition to a 26 °C control condition. An effective in vitro antioxidant activity was observed for [D-Leu1]MC-LR against hydrosoluble radicals. As far as we know, this is the first in vitro record of the role of MC as antioxidant. In addition, a significant increase in cellular biomass was observed under 26 °C in cultures with [D-Leu1]MC-LR supplementation in coincidence with a significant decrease of reactive species. For cultures at 29 °C, the antioxidant role of toxins was inconclusive probably due to the presence of different reactive species generated during the experiment. Thus, MC could scavenge certain reactive species associated with the antioxidant role of CAT or the OH content by SOD activity (not measured) and then CAT activity could be lower in the presence of MC. Reinforcing our hypothesis, the [D-Leu1]MC-LR consumption after 7 days was significantly higher in cells with [D-Leu1]MC-LR supplementation in both 26 °C and 29 °C.When the production of reactive species was controlled by the scavenger activity of antioxidants plus MC, cells avoided the potential oxidative damage and started with exponential growth.

Exploring the interaction of microcystin-LR with proteins and DNA.

The physiological role of microcystin-LR is still under discussion, and since binding of microcystin-LR to proteins different from their main cellular targets was described, we have performed experiments in order to explore this interaction. A non-specific interaction of microcystin-LR with a variety of soluble proteins in vitro is disrupted when using organic solvents such as methanol. The isoelectric point of proteins is not affected by their interaction with microcystin-LR, even though the presence of microcystin-LR alters the pool of peptides obtained by tryptic digestions. Under the conditions tested, microcystin-LR does not exhibit affinity for DNA. Although it is unlikely that the non-specific binding of microcystin-LR to proteins has a physiological meaning, one must be aware of the fact that determinations of the toxin extracted from any biological sample may be affected by the presence of proteins in the extracts. Consequently, we strongly recommend use organic solvents and to lyophilise the tissue samples to guarantee the accessibility of these organic solvents to microcystin-LR when performing experiments with tissue or cell extracts.

Global warming and hepatotoxin production by cyanobacteria: what can we learn from experiments?

Global temperature is expected to rise throughout this century, and blooms of cyanobacteria in lakes and estuaries are predicted to increase with the current level of global warming. The potential environmental, economic and sanitation repercussions of these blooms have attracted considerable attention among the world's scientific communities, water management agencies and general public. Of particular concern is the worldwide occurrence of hepatotoxic cyanobacteria posing a serious threat to global public health. Here, we highlight plausible effects of global warming on physiological and molecular changes in these cyanobacteria and resulting effects on hepatotoxin production. We also emphasize the importance of understanding the natural biological function(s) of hepatotoxins, various mechanisms governing their synthesis, and climate-driven changes in food-web interactions, if we are to predict consequences of the current and projected levels of global warming for production and accumulation of hepatotoxins in aquatic ecosystems.

Investigation of microcystin congener-dependent uptake into primary murine neurons.

BACKGROUND: Contamination of natural waters by toxic cyanobacteria is a growing problem worldwide, resulting in serious water pollution and human health hazards. Microcystins (MCs) represent a group of > 80 cyclic heptapeptides, mediating cytotoxicity via specific protein phosphatase (PP) inhibition at equimolar concentrations (comparable toxicodynamics). Because of the structure and size of MCs, active uptake into cells occurs via organic anion-transporting polypeptides (OATP/Oatp), as confirmed for liver-specific human OATP1B1 and OATP1B3, mouse Oatp1b2 (mOatp1b2), skate Oatp1d1, and the more widely distributed OATP1A2 expressed, for example, at the blood-brain barrier. Tissue-specific and cell-type-specific expression of OATP/Oatp transporters and specific transport of MC congeners (toxicokinetics) therefore appear prerequisite for the reported toxic effects in humans and other species upon MC exposure. Beyond hepatotoxicity induced by the MC-LR congener, the effects of other MC congeners, especially neuronal uptake and toxicity, are unknown. OBJECTIVES: In this study we examined the expression of mOatps and the uptake of congeners MC-LR, MC-LW, and MC-LF in primary murine neurons. METHODS: Intracellular MC accumulation was indicated indirectly via uptake inhibition experiments and directly confirmed by Western blot analysis and a PP inhibition assay. Neuronal mOatp expression was verified at the mRNA and protein level. RESULTS: MCs can cross neuronal cell membranes, with a subsequent decrease of PP activity. Of 15 mOatps, 12 were expressed at the mRNA level, but we found detectable protein levels for only two: mOatp1a5 (Slco1a5) and the known MC-LR transporter mOatp1b2 (Slco1b2). CONCLUSIONS: These data suggest mOatp-mediated uptake of MC congeners into neurons, thus corroborating earlier assumptions of the neurotoxic potential of MCs.

Structural characterization of in vitro and in vivo intermediates on the loading module of microcystin synthetase.

The microcystin family of toxins is the most common cause of hepatotoxicity associated with water blooms of cyanobacterial genera. The biosynthetic assembly line producing the toxic cyclic peptide, microcystin, contains an adenylation-peptidyl carrier protein didomain (A-PCP) at the N-terminus of the initiator module McyG (295 kDa) that has been postulated to activate and load the starter unit phenylacetate for formation of the unusual aromatic beta-amino acid residue, Adda, before subsequent extension. Characterization of the McyG A-PCP didomain (78 kDa) using ATP-PP i exchange assays and mass spectrometry revealed that assorted phenylpropanoids are preferentially activated and loaded onto the PCP carrier domain rather than phenylacetate itself. For the first time, thioesters formed in vivo were detected directly using large molecule mass spectrometry. Additionally substrates were cleaved using a type II thioesterase for structural elucidation by small molecule mass spectrometry. Unprecedented features of the McyG A-PCP didomain include the in vivo acylation of the holo PCP with exogenous and endogenous substrates, along with the ability of the apo protein to retain the acyl-AMP intermediate during affinity purification. These results imply that phenylpropanoids are preferentially loaded onto the McyG PCP; however one carbon must be excised following extension of the starter unit with malonyl-CoA in order to generate the expected polyketide chain which leads us to ponder the novel biochemistry by which this occurs.