Cyanobacterial occurrence and detection of microcystin by planar chromatography in surface water of Billings and Guarapiranga Reservoirs, SP, Brazil

Billings and Guarapiranga Reservoirs were deeply affected by environmental disturbances, which more evident consequence are the cyanobacterial blooms. Microcystins are the most common cyanotoxin in freshwaters and more than 70 types are known. Different methods for microcystins analysis in water can be used, among which ELISA and HPLC are the most frequently employed. However, less sophisticated and more economic methods can also be used. This is the case of planar chromatography (thin-layer chromatography) method previously used in cyanotoxins purification but gradually replaced by others. Posterior optimization of the microcystin chromatography conditions and because of its simplicity, rapidity, efficiency and low cost, this method is again considered an option for the analysis of microcystins and nodularins. Considering the importance of Billings and Guarapiranga Reservoirs for drinking water supplies and the few scientific data about cyanobacteria and cyanotoxins in these water bodies, the aims of this work are to analyze the biodiversity of cyanobacteria in the Billings and Guarapiranga Reservoirs and the detection of dissolved microcystins in the water. It was possible to identify 17 species of cyanobacteria, 9 of them being potentially toxic. In Billings Reservoir Microcystis aeruginosa (Kützing) Kützing and Cylindrospermopsis raciborskii (Woloszynska) Seenayya & Subba Raju are the most common species, while in Guarapiranga Reservoir only M. aeruginosa was considered as a common species. Microcystins were detected in all Billings Reservoir samples and in only one sample from Guarapiranga Reservoir.

Effects of microcystin-LR in isolated perfused rat kidney

Microcystin is a hepatotoxic peptide which inhibits protein phosphatase types 1 and 2A. The objective of the present study was to evaluate the physiopathologic effects of microcystin-LR in isolated perfused rat kidney. Adult Wistar rats (N = 5) of both sexes (240-280 g) were utilized. Microcystin-LR (1 µg/ml) was perfused over a period of 120 min, during which samples of urine and perfusate were collected at 10-min intervals to determine the levels of inulin, sodium, potassium and osmolality. We observed a significant increase in urinary flow with a peak effect at 90 min (control (C) = 0.20 ± 0.01 and treated (T) = 0.32 ± 0.01 ml g-1 min-1, P<0.05). At 90 min there was a significant increase in perfusate pressure (C = 129.7 ± 4.81 and T = 175.0 ± 1.15 mmHg) and glomerular filtration rate (C = 0.66 ± 0.07 and T = 1.10 ± 0.04 ml g-1 min-1) and there was a significant reduction in fractional sodium tubular transport at 120 min (C = 78.6 ± 0.98 and T = 73.9 ± 0.95%). Histopathologic analysis of the perfused kidneys showed protein material in the urinary space, suggestive of renal toxicity. These data demonstrate renal vascular, glomerular and urinary effects of microcystin-LR, indicating that microcystin acts directly on the kidney by probable inhibition of protein phosphatases.

Microcystin-LR in Brazilian Aquaculture Production Systems

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Degradation of [D-Leu]-Microcystin-LR by solar heterogeneous photocatalysis (TiO2)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluation of the photoelectrocatalytic method for oxidizing chloride and simultaneous removal of microcystin toxins in surface waters

The production of chlorine was investigated in the photoelectrocatalytic oxidation of a chloride-containing solution using a TiO(2) thin-film electrode biased at current density from 5 to 50 mA cm(-2) and illuminated by UV light. Such parameters as chloride concentrations from 0.001 to 0.10 mol L(-1), pH 2-12, and interfering salts were varied in this study in order to determine their effect on this oxidation process. At an optimum condition this photoelectrocatalytic method can produce active chlorine at levels compatible to water disinfections processes using a chloride concentration higher than 0.010 mol L(-1) at a pH of 4 and a current density of 30 mA cm(-2). The method was successfully applied to treat surface water collected from a Brazilian river. After 150 min of photoelectrocatalytic oxidation, we obtained a 90% reduction in total organic carbon removal, a 100% removal of turbidity, a 93% decrease in colour and a chemical oxygen demand (COD) removal of around 96% (N=3). The proposed technology based on photoelectrocatalytic oxidation was also tested in treating 250 mL of a solution containing 0.05 mol L(-1) NaCl and 50 mu g L(-1) of Microcystin aeruginosa. The bacteria is completely removed after 5 min of photoelectrocatalysis following an initial rate constant removal of -0.260 min(-1), suggesting that the present method could be considered as a promising alternative to chlorine-based disinfections. (C) 2008 Elsevier Ltd. All rights reserved.

Microcystin-producing genotypes from cyanobacteria in Brazilian reservoirs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Co-occurrence of microcystin and microginin congeners in Brazilian strains of Microcystis sp.

Species of Microcystis are the most common bloom-forming cyanobacteria in several countries. Despite extensive studies regarding the production of bioactive cyanopeptides in this genus, there are limited data on isolated strains from Brazil. Three Microcystis sp. strains were isolated from the Salto Grande Reservoir (LTPNA01, 08 and 09) and investigated for the presence of mcy genes, microcystins and other cyanopeptides. Microcystin and microginin production was confirmed in two isolates using high-resolution tandem mass spectrometry after electrospray ionization (ESI-Q-TOF), and the structures of two new microginin congeners were proposed (MG756 Ahda-Val-Leu-Hty-Tyr and MG770 MeAhda-Val-Leu-Hty-Tyr). The biosynthesis profile of the identified cyanopeptides was evaluated at different growth phases via a newly developed HPLC-UV method. Results demonstrated no substantial differences in the production of microcystins and microginins after data normalization to cell quota, suggesting a constitutive biosynthesis. This study represents the first confirmed co-production of microginins and microcystins in Brazilian strains of Microcystis sp. and highlights the potential of Brazilian cyanobacteria as a source of natural compounds with pharmaceutical interest.

Microcystin-producing genotypes from cyanobacteria in Brazilian reservoirs

The aim of this study was to evaluate the use of new oligonucleotide primers (mcyB-F/R, mcyB-F/R-A, and mcyB-F/R-B) designed from Brazilian cyanobacteria for the detection of microcystin-producing genotypes in 27 environmental samples from water reservoirs and 11 strains of Microcystis. Microcystins were found using HPLC in all 11 strains and 19 of the environmental samples. The new oligonucleotide primers amplified fragments of microcystin-producing genes, including the eight environmental samples in which no microcystins were detected by HPLC, but which presented amplified fragments, thereby demonstrating the existence of microcystin-producing genes. The new oligonucleotide primers exhibited better specificity when used with environmental samples and were more reliable in comparison with those described in the literature (mcyB-FAA/RAA and mcyA-Cd/FR), which generate false-negative results. The better performance of these new oligonucleotide primers underline the need for designing molecular markers that are well fitted to the regional biological diversity. As this is a fast predictive technique for determining the presence or absence of microcystins, it could be used either alone or in conjunction with other techniques, such as the screening of samples to be sent for quantitative toxicological analysis using HPLC, thereby reducing monitoring cost and time. (c) 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

Degradation of [D-Leu]-Microcystin-LR by solar heterogeneous photocatalysis (TiO2)

The present study investigates the use of solar heterogeneous photocatalyis (TiO2) for the destruction of [D-Leu]-Microcystin-LR, powerful toxin of widespread occurrence within cyanobacteria blooms. We extracted [D-Leu]-Microcystin-LR from a culture of Microcystis spp. and used a flat plate glass reactor coated with TiO2 (Degussa, P25) for the degradation studies. The irradiance was measured during the experiments with the aid of a spectroradiometer. After the degradation experiments, toxin concentrations were determined by HPLC and mineralization by TOC analyses. Acute and chronic toxicities were, quantified using mice and phosphatase inhibition in vitro assays, respectively. According to the performed experiments, 150 min were necessary to reduce the toxin concentration to the WHO's guideline for drinking water (from 10 to 1 mu g L-1) and to mineralize 90% of the initial carbon content. Another important finding is that solar heterogeneous photocatalysis was a destructive process indeed, not only for the toxin, but also for the other extract components and degradation products generated. Moreover, toxicity tests using mice have shown that the acute effect caused by the initial sample was removed. However, tests using the phosphatase enzyme indicated that it may be formed products capable of inducing chronic effects on mammals. The performed experiments indicate the feasibility of using solar heterogeneous photocatalysis for treating contaminated water with [D-Leu]-Microcystin-LR, not only due to its destruction, but also to the significant removal of organic matter and acute toxicity that can be achieved. (C) 2012 Elsevier Ltd. All rights reserved.

Biomonitoring of Microcystin and Aflatoxin Co-Occurrence in Aquaculture Using Immunohistochemistry and Genotoxicity Assays

This work investigated the effects of co-occurring aflatoxin B-1 (AFB(1)) and microcystin (MC) in aquaculture, using immunohistochemistry and genotoxicity methods. Tilapia (Oreochromis niloticus) were exposed to AFB(1) by intraperitoneal and MC (cell extract of Microcystis aeruginosa) by intraperitoneal and immersion routes. The interaction of MC-AFB(1) was evaluated co-exposing the intraperitoneal doses. Blood samples were collected after 8, 24, and 48h to analyze the micronucleus frequency and comet score. The interaction of MC-AFB(1) showed a synergic mutagenic response by higher micronucleus frequency of co-exposed group. A slight genotoxic synergism was also observed in the comet score. Immunohistochemistry detected MC in al lthe fish liver tissues exposed to MC by intraperitoneal route, and only the immersed group with the highest dose of MC showed a positive response. Although MC was non-detectable in the edible muscle, the combination of immunohistochemistry with genotoxicity assay was an attractive biomonitoring tool in aquaculture, where the animals were frequently exposed to co-occurring synergic hazards.

Convergent evolution of [D-Leucine1] microcystin-LR in taxonomically disparate cyanobacteria

Abstract Background Many important toxins and antibiotics are produced by non-ribosomal biosynthetic pathways. Microcystins are a chemically diverse family of potent peptide toxins and the end-products of a hybrid NRPS and PKS secondary metabolic pathway. They are produced by a variety of cyanobacteria and are responsible for the poisoning of humans as well as the deaths of wild and domestic animals around the world. The chemical diversity of the microcystin family is attributed to a number of genetic events that have resulted in the diversification of the pathway for microcystin assembly. Results Here, we show that independent evolutionary events affecting the substrate specificity of the microcystin biosynthetic pathway have resulted in convergence on a rare [D-Leu1] microcystin-LR chemical variant. We detected this rare microcystin variant from strains of the distantly related genera Microcystis, Nostoc, and Phormidium. Phylogenetic analysis performed using sequences of the catalytic domains within the mcy gene cluster demonstrated a clear recombination pattern in the adenylation domain phylogenetic tree. We found evidence for conversion of the gene encoding the McyA2 adenylation domain in strains of the genera Nostoc and Phormidium. However, point mutations affecting the substrate-binding sequence motifs of the McyA2 adenylation domain were associated with the change in substrate specificity in two strains of Microcystis. In addition to the main [D-Leu1] microcystin-LR variant, these two strains produced a new microcystin that was identified as [Met1] microcystin-LR. Conclusions Phylogenetic analysis demonstrated that both point mutations and gene conversion result in functional mcy gene clusters that produce the same rare [D-Leu1] variant of microcystin in strains of the genera Microcystis, Nostoc, and Phormidium. Engineering pathways to produce recombinant non-ribosomal peptides could provide new natural products or increase the activity of known compounds. Our results suggest that the replacement of entire adenylation domains could be a more successful strategy to obtain higher specificity in the modification of the non-ribosomal peptides than point mutations.

Biomonitoring of microcystin and aflatoxin co-occurrence in aquaculture using immunohistochemistry and genotoxicity assays

This work investigated the effects of co-occurring aflatoxin B1 (AFB1) and microcystin (MC) in aquaculture, using immunohistochemistry and genotoxicity methods. Tilapia (Oreochromis niloticus) were exposed to AFB1 by intraperitoneal and MC (cell extract of Microcystis aeruginosa) by intraperitoneal and immersion routes. The interaction of MC-AFB1 was evaluated co-exposing the intraperitoneal doses. Blood samples were collected after 8, 24, and 48h to analyze the micronucleus frequency and comet score. The interaction of MC-AFB1 showed a synergic mutagenic response by higher micronucleus frequency of co-exposed group. A slight genotoxic synergism was also observed in the comet score. Immunohistochemistry detected MC in al lthe fish liver tissues exposed to MC by intraperitoneal route, and only the immersed group with the highest dose of MC showed a positive response. Although MC was non-detectable in the edible muscle, the combination of immunohistochemistry with genotoxicity assay was an attractive biomonitoring tool in aquaculture, where the animals were frequently exposed to co-occurring synergic hazards.

Biodegradation of the cyanobacterial toxin microcystin LR in natural water and biologically active slow sand filters

A bacterium (MJ-PV) previously demonstrated to degrade the cyanobacterial toxin microcystin LR, was investigated for bioremediation applications in natural water microcosms and biologically active slow sand filters. Enhanced degradation of microcystin LR was observed with inoculated (1 x 10(6) cell/mL) treatments of river water dosed with microcystin LR (> 80% degradation within 2 days) compared to uninoculated controls. Inoculation of MJ-PV at lower concentrations (1 x 10(2)-1 x 10(5)cells/mL) also demonstrated enhanced microcystin LR degradation over control treatments. Polymerase chain reactions (PCR) specifically targeting amplification of 16S rDNA of MJ-PV and the gene responsible for initial degradation of microcystin LR (mlrA) were successfully applied to monitor the presence of the bacterium in experimental trials. No amplified products indicative of an endemic MJ-PV population were observed in uninoculated treatments indicating other bacterial strains were active in degradation of microcystin LR, Pilot scale biologically active slow sand filters demonstrated degradation of microcystin LR irrespective of MJ-PV bacterial inoculation. PCR analysis detected the MJ-PV population at all locations within the sand filters where microcystin degradation was measured. Despite not observing enhanced degradation of microcystin LR in inoculated columns compared to uninoculated column, these studies demonstrate the effectiveness of a low-technology water treatment system like biologically active slow sand filters for removal of microcystins from reticulated water supplies. Crown Copyright (c) 2006 Published by Elsevier Ltd. All rights reserved.

Hydroxyl radical generation by cactus-like copper oxide nanoporous carbon catalysts for microcystin-LR environmental remediation

Copper oxide supported on nanoporous activated carbon (CuO-NPAC) is reported for the aqueous phase catalytic degradation of cyanotoxin microcystin-LR (MC-LR). The loading and spatial distribution of CuO throughout the NPAC matrix strongly influence the catalytic efficiency. CuO-NPAC synthesis was optimized with respect to the copper loading and thermal processing, and the physicochemical properties of the resulting materials were characterized by XRD, BET, TEM, SEM, EPR, TGA, XPS and FT-IR spectroscopy. EPR spin trapping and fluorescence spectroscopy showed in situ ˙OH formation via H2O2 over CuO-NPAC as the catalytically relevant oxidant. The impact of reaction conditions, notably CuO-NPAC loading, H2O2 concentration and solution pH, is discussed in relation to the reaction kinetics for MC-LR remediation.