In vitro and in silico assessment of cytotoxicity and chromosome instability induced by saxitoxin in human derived neural cell line.

In freshwater, saxitoxins (STX) are produced by different cyanobacteria genera, including Raphidiopsis. Data regarding cytogenotoxicity effects of STX on human cells are scarse, this merit further studies of its toxicology. This study assessed the cytotoxicity and the chromosome instability of STX on SHSY-5Y human cell line. The CBMN assay allows the detection of chromosome breaks and abnormal chromosomal segregation. Additionally, in silico systems biology approach, used to search for known and predicted interaction networks, was applied to study the interactions between STX and SHSY-5Y cellular components. The results of the CBMN assay demonstrated that STX concentrations of 2.5 - 10 µg/L induced cytostasis and chromosome instability in a dose-response relationship. Apoptosis was detected after exposure of SHSY-5Y cultured cells to STX concentration of 10 µg/L. The results of the systems biology analysis revealed the interaction of STX with proteins related with acetylcoline pathway, cell cycle regulation and apoptosis. Furthermore, combining the in vitro and in silico approachs, it was possible to suggest a mechanism of action of STX in SHSY-5Y cells. Overall, the data demonstrated the cytotoxicity and mutagenicity of environmentally relevant concentrations of STX. These results should be considered when setting up guidelines for monitoring STX in water supply.

The effects of Microcystis aeruginosa cells lysate containing microcystins on physiological and molecular responses in the nematode Caenorhabditis elegans.

Microcystins (MCs) are potent toxins produced by environmental cyanobacterial blooms. The present study evaluated the effects of a Microcystis aeruginosa cyanobacterial lysate containing 0.1, 1, and 10 µg L-1 MC-LR equivalent in the C. elegans Bristol N2 wild-type and the effects caused by equivalent concentrations of a MC-LR standard. The lysate was prepared from a culture of toxic strain (RST9501) originated from the Patos Lagoon Estuary (RS, Brazil). The minimal concentration necessary to cause significant effects in C. elegans under exposure to M. aeruginosa lysate or to MC-LR standard were, respectively, 10 and 0.1 µg L-1 MC-LR equivalent for growth and 10 and 1 µg L-1 MC-LR equivalent for fertility. Reproduction (ie, brood size) was only affected by the exposure to 10 µg L-1 MC-LR standard and was not affected by the lysate. The nematodes that were exposed to lysate containing 1 µg L-1 MC-LR equivalent or MC-LR were also analyzed for pharyngeal pumping and gene expression using RT-qPCR. The worms' rhythmic contractions of the pharynx were similarly affected by the lysate containing 1 µg L-1 of MC-LR equivalent and the MC-LR standard. The MC-LR standard caused down-regulation of genes related to growth (daf-16), fertility (spe-10), and biotransformation (gst-2). This is the first study to evaluate the effects of a toxic cyanobacterial lysate using the C. elegans model. This study suggests the organism as a potential biotest to evaluate toxicity of natural waters containing M. aeruginosa cells and to environmental risk assessment associated to cyanobacterial bloom events.

Environmental Variability and Cyanobacterial Blooms in a Subtropical Coastal Lagoon: Searching for a Sign of Climate Change Effects.

Cyanobacterial blooms in marine and freshwater environments may be favored by shifts in physical water column parameters due to warming under climate change. The Patos Lagoon (PL), a subtropical coastal environment in southern Brazil, is known for recurrent blooms of Microcystis aeruginosa complex (MAC). Here, we analyze the variability of these blooms and their relation to changes in wind direction and speed, rainfall and freshwater run-off from 2000 to 2017. Also, we discuss both longer time-series of air temperature and rainfall and a review of local studies with microcystins produced by these noxious species. Since the 1980s, MAC blooms were associated to negative anomalies in annual precipitation that occur during La Niña periods and, in the last years (2001-2014), accompanied by a trend in low river discharge. MAC blooms were conspicuous from December to March, i.e., austral summer, with massive patches seen in satellite images as for 2017. We suggest that low rainfall and run-off years under NE wind-driven hydrodynamics might accumulate MAC biomass in the west margin of the PL system. In contrast, a positive, long-term trend in precipitation (from 1950 to 2016; slope = 3.9868 mm/yr, p < 0.05) should imply in high river discharge and, consequently, advection of this biomass to the adjacent coastal region. Due to the proximity to urban areas, the blooms can represent recreational and economic hazards to the region.

Growth Characteristics of an Estuarine Heterocystous Cyanobacterium.

A new estuarine filamentous heterocystous cyanobacterium was isolated from intertidal sediment of the Lagoa dos Patos estuary (Brazil). The isolate may represent a new genus related to Cylindrospermopsis. While the latter is planktonic, contains gas vesicles, and is toxic, the newly isolated strain is benthic and does not contain gas vesicles. It is not known whether the new strain is toxic. It grows equally well in freshwater, brackish and full salinity growth media, in the absence of inorganic or organic combined nitrogen, with a growth rate 0.6 d-1. Nitrogenase, the enzyme complex responsible for fixing dinitrogen, was most active during the initial growth phase and its activity was not different between the different salinities tested (freshwater, brackish, and full salinity seawater). Salinity shock also did not affect nitrogenase activity. The frequency of heterocysts was high, coinciding with high nitrogenase activity during the initial growth phase, but decreased subsequently. However, the frequency of heterocysts decreased considerably more at higher salinity, while no change in nitrogenase activity occurred, indicating a higher efficiency of dinitrogen fixation. Akinete frequency was low in the initial growth phase and higher in the late growth phase. Akinete frequency was much lower at high salinity, which might indicate better growth conditions or that akinete differentiation was under the same control as heterocyst differentiation. These trends have hitherto not been reported for heterocystous cyanobacteria but they seem to be well fitted for an estuarine life style.

Biodegradation of [D-Leu(1)] microcystin-LR by a bacterium isolated from sediment of Patos Lagoon estuary, Brazil.

BACKGROUND: Toxic cyanobacterial blooms are recurrent in Patos Lagoon, in southern Brazil. Among cyanotoxins, [D-Leu(1)] microcystin-LR is the predominant variant whose natural cycle involves water and sediment compartments. This study aimed to identify and isolate from sediment a bacterial strain capable of growing on [D-Leu(1)] microcystin-LR. Sediment and water samples were collected at two distinct aquatic spots: close to the Oceanographic Museum (P1), in Rio Grande City, and on São Lourenço Beach (P2), in São Lourenço do Sul City, southern Brazil. METHODS: [D-Leu(1)] microcystin-LR was isolated and purified from batch cultures of Microcystis aeruginosa strain RST9501. Samples of water and sediment from Rio Grande and São Lourenço do Sul were collected. Bacteria from the samples were allowed to grow in flasks containing solely [D-Leu(1)] microcystin-LR. This strain named DMSX was isolated on agar MSM with 8 g L(-1) glucose and further purified on a cyanotoxin basis growth. Microcystin concentration was obtained by using the ELISA immunoassay for microcystins whereas bacterial count was performed by epifluorescence microscopy. The genus Pseudomonas was identified by DNA techniques. RESULTS: Although several bacterial strains were isolated from the samples, only one, DMXS, was capable of growing on [D-Leu(1)] microcystin-LR. The phylogenetic analysis of the 16S rRNA gene from DMXS strain classified the organism as Pseudomonas aeruginosa. DMXS strain incubated with [D-Leu(1)] microcystin-LR lowered the amount of toxin from 1 µg.L(-1) to < 0.05 µg.L(-1). Besides, an increase in the bacterial count-from 71 × 10(5) bacteria.mL(-1) to 117 × 10(5) bacteria.mL(-1)-was observed along the incubation. CONCLUSIONS: The use of bacteria isolated from sediment for technological applications to remove toxic compounds is viable. Studies have shown that sediment plays an important role as a source of bacteria capable of degrading cyanobacterial toxins. This is the first Brazilian report on a bacterium-of the genus Pseudomonas-that can degrade [D-Leu(1)] microcystin-LR, the most frequent microcystin variant in Brazilian freshwaters.

Biodegradation of [D-Leu1] microcystin-LR by a bacterium isolated from sediment of Patos Lagoon estuary, Brazil

Background Toxic cyanobacterial blooms are recurrent in Patos Lagoon, in southern Brazil. Among cyanotoxins, [D-Leu1] microcystin-LR is the predominant variant whose natural cycle involves water and sediment compartments. This study aimed to identify and isolate from sediment a bacterial strain capable of growing on [D-Leu1] microcystin-LR. Sediment and water samples were collected at two distinct aquatic spots: close to the Oceanographic Museum (P1), in Rio Grande City, and on São Lourenço Beach (P2), in São Lourenço do Sul City, southern Brazil. Methods [D-Leu1] microcystin-LR was isolated and purified from batch cultures of Microcystis aeruginosastrain RST9501. Samples of water and sediment from Rio Grande and São Lourenço do Sul were collected. Bacteria from the samples were allowed to grow in flasks containing solely [D-Leu1] microcystin-LR. This strain named DMSX was isolated on agar MSM with 8 g L1 glucose and further purified on a cyanotoxin basis growth. Microcystin concentration was obtained by using the ELISA immunoassay for microcystins whereas bacterial count was performed by epifluorescence microscopy. The genus Pseudomonas was identified by DNA techniques. Results Although several bacterial strains were isolated from the samples, only one, DMXS, was capable of growing on [D-Leu1] microcystin-LR. The phylogenetic analysis of the 16S rRNA gene from DMXS strain classified the organism as Pseudomonas aeruginosa. DMXS strain incubated with [D-Leu1] microcystin-LR lowered the amount of toxin from 1 g.L1 to 0.05 g.L1. Besides, an increase in the bacterial countfrom 71×105 bacteria.mL1 to 117×105 bacteria.mL1was observed along the incubation. Conclusions The use of bacteria isolated from sediment for technological applications to remove toxic compounds is viable. Studies have shown that sediment plays an important role as a source of bacteria capable of degrading cyanobacterial toxins. This is the first Brazilian report on a bacteriumof the genus Pseudomonasthat can degrade [D-Leu1] microcystin-LR, the most frequent microcystin variant in Brazilian freshwaters.

Biodegradation of [D-Leu1] microcystin-LR by a bacterium isolated from sediment of Patos Lagoon estuary, Brazil

Background: Toxic cyanobacterial blooms are recurrent in Patos Lagoon, in southern Brazil. Among cyanotoxins, [D-Leu1] microcystin-LR is the predominant variant whose natural cycle involves water and sediment compartments. This study aimed to identify and isolate from sediment a bacterial strain capable of growing on [D-Leu1] microcystin-LR. Sediment and water samples were collected at two distinct aquatic spots: close to the Oceanographic Museum (P1), in Rio Grande City, and on São Lourenço Beach (P2), in São Lourenço do Sul City, southern Brazil. Methods: [D-Leu1] microcystin-LR was isolated and purified from batch cultures of Microcystis aeruginosastrain RST9501. Samples of water and sediment from Rio Grande and São Lourenço do Sul were collected. Bacteria from the samples were allowed to grow in flasks containing solely [D-Leu1] microcystin-LR. This strain named DMSX was isolated on agar MSM with 8 g L−1 glucose and further purified on a cyanotoxin basis growth. Microcystin concentration was obtained by using the ELISA immunoassay for microcystins whereas bacterial count was performed by epifluorescence microscopy. The genus Pseudomonas was identified by DNA techniques. Results; Although several bacterial strains were isolated from the samples, only one, DMXS, was capable of growing on [D-Leu1] microcystin-LR. The phylogenetic analysis of the 16S rRNA gene from DMXS strain classified the organism as Pseudomonas aeruginosa. DMXS strain incubated with [D-Leu1] microcystin-LR lowered the amount of toxin from 1 μg.L−1 to < 0.05 μg.L−1. Besides, an increase in the bacterial count–from 71 × 105 bacteria.mL−1 to 117 × 105 bacteria.mL−1–was observed along the incubation. Conclusions: The use of bacteria isolated from sediment for technological applications to remove toxic compounds is viable. Studies have shown that sediment plays an important role as ...

Expression and activity of glutathione S-transferases and catalase in the shrimp Litopenaeus vannamei inoculated with a toxic Microcystis aeruginosa strain.

Microcystin (MC) produced during cyanobacteria blooms is notably toxic to human and wildlife. Conjugation with reduced glutathione (GSH) by glutathione S-transferase (GST) and the antioxidant enzymes defenses (e.g. catalase, CAT) are important biochemical defense mechanisms against MCs toxicity. We investigated the enzymatic activity of CAT and GST and the gene expression levels of CAT and eight GST isoforms in the hepatopancreas of the globally farmed shrimp Litopenaeus vannamei 48-h after injection with a sub-lethal dose of 100 µg kg⁻¹ of a toxic Microcystis aeruginosa extract. MCs caused up-regulation for GSTΩ, µ and a MAPEG isoform, by 12-, 2.8- and 1.8-fold, respectively, and increases in the total GST enzyme activity and CAT enzyme activity. The study points to the importance of further characterization for the L. vannamei GST isoforms and GST/CAT post-translational regulation processes to better understand the key mechanisms involved in the shrimp's defense against MC exposure.

Chemoprotection of lipoic acid against microcystin-induced toxicosis in common carp (Cyprinus carpio, Cyprinidae).

This paper evaluated the chemoprotective effect of lipoic acid (LA) against microcystin (MC) toxicity in carp Cyprinus carpio. To determine the LA dose and the time necessary for the induction of three different classes (alpha, mu and pi) of glutathione S-transferase (GST) gene transcription, carp were i.p. injected with 40mg/kg lipoic acid solution. A group was killed 24h after the first i.p. injection (condition 1); another group received two i.p. injections with a 24h of interval between each one and was killed 48h after the first injection (condition 2) and a third group received one i.p. injection and was killed 48h latter (condition 3). Results showed that LA was effective in promoting an increase in GSTs gene transcription in liver only in the condition 2. A second experiment was done, where carp pre-treated with LA (condition 2) were gavaged twice with a 24h interval with 50µg MC/kg. Ninety-six hours after experiment beginning, carp were killed, and organs were dissected. Results of GST activity in liver and brain suggest that LA can be a useful chemoprotection agent against MC induced toxicity, stimulating detoxification through the increment of GST activity (brain) or through reversion of GST inhibition (liver).

Biodegradation of microcystins by aquatic Burkholderia sp. from a South Brazilian coastal lagoon.

Biological degradation of cyanobacterial hepatotoxin microcystins in estuarine and coastal water samples from the Patos Lagoon estuarine system, a coastal lagoon situated at the southernmost region of Brazil, was observed. Samples of natural surface water were spiked with purified and semi-purified microcystins (MC-LR and [D-Leu(1)]MC-LR) and their concentrations were monitored by HPLC analysis. After 15 days, the toxins were no longer detectable and after 43 days less than 90% of the initial concentration added to the samples was detected by ELISA. The average degradation rates and the exponential decay rate constants from inside and outside of the estuary were similar. A microcystin degradative bacterium was isolated from the estuarine region. Partial sequence of the 16S rDNA showed a 96% homology with the Burkholderia genus. This genus belongs to the beta subdivision on proteobacteria. This is the first report showing the genus Burkholderia as a cyanobacterial toxin degrader.

Cyanobacterial blooms in estuarine ecosystems: characteristics and effects on Laeonereis acuta (Polychaeta, Nereididae).

In January of 2003, a cyanobacterial bloom in the Patos' Lagoon (Southern Brazil) (32 degrees 05'S-52 degrees 12'W) was observed. Water samples were taken to identify the composition and abundance of the bloom, as well as the occurrence of toxins. The effects of this occurrence on the estuarine worm Laeonereis acuta (Polychaeta, Nereididae) was also evaluated. Predominance of cyanobacteria, particularly Anabaena trichomes ( approximately 2.5.10(6) individuals per liter) was observed, and low concentrations of microcystins and anticholinesterasic toxins were detected. Augmented levels of lipid hydroperoxides (LPO) and glutathione-S-transferase activity, and lowering of total protein content were also observed in organisms collected during the bloom event. Although non-toxic, the cyanobacterial bloom could augment the cycle of hyper-oxygenation and hypoxia in the water. During hyperoxia, L. acuta, an oxyconformer, should consume more oxygen, thus augmenting the rate of reactive oxygen species generation. A repeated cycle of hyper-oxygenation and hypoxia would finally induce oxidative stress, as evidenced by the high levels of LPO and glutathione-S-transferase activity.