The role of glutathione metabolism in tolerance of tobacco BY-2 suspension cells to microcystin-RR

This study was undertaken to investigate the role of the glutathione-involved detoxifying mechanism in defending the tobacco BY-2 suspension cells against microcystin-RR (MC-RR). Analysis showed that exposure of the cells to different concentrations of MC-RR (0.1, 1 and 10 mu g/mL) for 0-6 days resulted in a time and concentration-dependent decrease in cell viability and increase in reactive oxygen species (ROS) content. Reduced glutathione (GSH) and total glutathione (tGSH) content as well as glutathione reductase (GR), glutathione peroxidase (GPX) and glutathione-S-transferase (GST) activities significantly increased after 3-4 days exposure in the highest two concentration treated groups, while decreased until reaching the control values except for GPX at day 6. Oxidized glutathione (GSSG) content markedly increased compared with control in high concentration MC-RR treated group after 6 days exposure. The GSH/GSSG ratio was much higher than control in 10 mu g/mL MC-RR treated group at day 4, but after 6 days exposure, the ratios in all treated groups were lower than that of the control group.

A novel chemiluminescent immunoassay for microcystin (MC) detection based on gold nanoparticles label and its application to MC analysis in aquatic environmental samples

A novel chemiluminescent immunoassay method based on gold nanoparticles was developed for the detection of microcystins (MCs). The immunoassay included three main steps: indirect competitive immunoreaction, oxidative dissolution of gold nanoparticles, and indirect determination for MCs with Au3+-catalysed luminol chemiluminesent system. The method has a wide working range (0.05-10 mu g L-1, r(2) = 0.9914), the limit of detection was determined to be 0.024 mu g L-1, which is much lower than the World Health Organization's proposed guidelines (1 mu g L-1) for drinking-water. The proposed method was applied to MC analysis in natural water and fish tissue samples, and most results in the proposed method were in agreement with the conventional indirect competitive enzyme-linked immunosorbent assay method, which indicated that the new chemiluminescent immunoassay was sensitive, reliable, and suitable for MC analysis in natural water and fish tissue samples.

Reduction in microcystin concentrations in large and shallow lakes: Water and sediment-interface contributions

Blooms of cyanobacteria, or blue-greens, are known to produce chemicals, such as microcystins, which can be toxic to aquatic and terrestrial organisms. Although previous studies have examined the fate of microcystins in freshwater lakes, primary elimination pathways and factors affecting degradation and loss have not been fully explained. The goal of the present study was to explore sources of algal toxins and investigate the distribution and biodegradation of microcystins in water and sediment through laboratory and field analyses. Water and sediment samples were collected monthly from several locations in Lake Taihu from February 2005 to January 2006. Samples were analyzed for the presence of microcystin. Water and sediment were also used in laboratory studies to determine microcystin degradation rates by spiking environmental samples with known concentrations of the chemical and observing concentration changes over time. Some water samples were found to efficiently degrade microcystins. Microcystin concentrations dropped faster in water collected immediately above lake sediment (overlying water). Degradation in sediments was higher than in water. Based on spatial distribution analyses of microcystin in Lake Taihu, higher concentrations (relative to water concentrations) of the chemical were found in lake sediments. These data suggest that sediments play a critical role in microcystin degradation in aquatic systems. The relatively low levels of microcystins found in the environment are most likely due to bacterial biodegradation. Sediments play a crucial role as a source (to the water column) of bio-degrading bacteria and as a carbon-rich environment for bacteria to proliferate and metabolize microcystin and other biogenic toxins produced by cyanobacteria. These, and other, data provide important information that may be applied to management strategies for improvement of water quality in lakes, reservoirs and other water bodies. (C) 2007 Elsevier Ltd. All rights reserved.

A laboratory study on risk assessment of microcystin-RR in cropland

The persistence time and risk of microcystin-RR (MC-RR) in cropland via irrigation were investigated under laboratory conditions. In order to evaluate the efficiency of the potential adsorption and biodegradation of MC-RR in cropland and the persistence time of MC-RR for crop irrigation, high performance liquid chromatography (HPLC) was used to quantify the amount of MC-RR in solutions. Our study indicated that MC-RR could be adsorbed and biodegraded in cropland soils. MC-RR at 6.5 mg/L could be completely degraded within 6 days with a lag phase of 1 - 2 days. In the presence of humic acid, the same amount of MC-RR could be degraded within 4 days without a lag phase. Accordingly, the persistence time of MC-RR in cropland soils should be about 6 days. This result also suggested the beneficial effects of the organic fertilizer utilization for the biodegradation of MC-RR in cropland soils. Our studies also demonstrated that MC-RR at low concentration (< 10 mu g/L) could accelerate the growth of plants, while high concentration of MC-RR (> 100 mu g/L) significantly inhibited the growth of plants. High sensitivity of the sprouting stage plants to MC-RR treatments as well as the strong inhibitory effects resulting from prolonged irrigation further indicated that this MC-RR growth-inhibition may vary with the duration of irrigation and life stage of the plants. (c) 2007 Published by Elsevier Ltd.

Effects of microcystin-RR on the antioxidant system of Bacillus subtilis

Microcystins are a kind of cyclic hepatotoxins produced by many cyanobacterial species. Many works have been done concerning, the toxic effects of microcystins on animals and plants. However, the reports about their effects on microbial cells are very limited. In the present paper, Bacillus subtilis (B. subtilis) was used to determine the dose- and time-effect of microcystin-RR, and the results showed that the activity of antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT) was significantly increased to that of control, when exposed to 5 or 10 mu g/ml microcystin-RR for 1 h. The contents of thiobarbituric acid-reactive sub-stances (TBARS) and glutathione (GSH) as well as glu-tathione reductase (GR) activity were obviously increased only when exposed to 10 mu g/ml microcystin-RR. For the time-effect of microcystin-RR on B. subtilis, the activities of antioxidant enzymes including SOD and CAT as well as GR activity and TBARS, GSH contents in B. subtilis were at first significantly increased, and then subsequently de-creased. These results suggested that microcystin-RR could induce the oxidative stress of B. subtilis for a short period. The antioxidant system protects B. subtilis from oxidative damage.

In situ studies on the distribution patterns and dynamics of microcystins in a biomanipulation fish - bighead carp (Aristichthys nobilis)

The distribution and dynamics of microcystins in various organs of the phytoplanktivorous bighead carp were studied monthly in Lake Taihu, which is dominated by toxic cyanobacteria. There was a good agreement between LC-MS and HPLC-UV determinations. Average recoveries of spiked fish samples were 63% for MC-RR and 71% for MC-LR. The highest MC contents in intestine, liver, kidney and spleen were 85.67, 2.83, 1.70 and 1.57 mu g g(-1) DW, respectively. MCs were much higher in mid-gut walls (1.22 mu g g(-1) DW) than in hind- and fore-gut walls (0.31 and 0.18 mu g g(-1) DW, respectively), suggesting the importance of mid-gut wall as major site for MC absorption. A cysteine conjugate of MC-LR was detected frequently in kidney. Among the muscle samples analyzed, 25% were above the provisional tolerable daily intake level by WHO. Bighead is strongly resistant to microcystins and can be used as biomanipulation fish to counteract cyanotoxin contamination in eutrophic waters. (c) 2006 Elsevier Ltd. All rights reserved.

Effects of nonylphenol on the growth and microcystin production of Microcystis strains

Both organic pollution and eutrophication are prominent environmental issues concerning water pollution in the world. It is important to reveal the effects of organic pollutants on algal growth and toxin production for assessing ecological risk of organic pollution. Since nonylphenol (NP) is a kind of persistent organic pollutant with endocrine disruptive effect which exists ubiquitously in environments, NP was selected as test compound in our study to study the relationship between NP stress and Microcystis growth and microcystin production. Our study showed that responses of toxic and nontoxic Microcystis aeruginosa to NP stress were obviously different. The growth inhibition test with NP on M. aeruginosa yielded effect concentrations EbC50 values within this range of 0.67-2.96 mg/L. The nontoxic M. aeruginosa strains were more resistant to NP than toxic strains at concentration above 1 mg/L. Cell growth was enhanced by 0.02-0.2 mg/L NP for both toxic and nontoxic strains, suggesting a hormesis effect of NP on M. aeruginosa. Both toxic and nontoxic strains tended to be smaller with increasing NP. But with the increased duration of the experiment, both the cell size and the growth rate began to resume, suggesting a quick adaptation of M. aeruginosa to adverse stress. NP of 0.05-0.5 mg/L significantly promoted microcystin production of toxic strain PCC7820, suggesting that NP might affect microcystin production of some toxic M. aeruginosa in the field. Our study showed that microcystin excretion was species specific that up to 75% of microcystins in PCC7820 were released into solution, whereas > 99% of microcystins in 562 remained in algal cells after 12 days' incubation. NP also significantly influenced microcystin release into cultural media. The fact that NP enhanced growth and toxin production of M. aeruginosa at low concentrations of 0.02-0.5 mg/L that might be possibly found in natural freshwaters implies that low concentration of NP may favor survival of M. aeruginosa in the field and may play a subtle role in affecting cyanobacterial blooms and microcystin production in natural waters. (c) 2006 Elsevier Inc. All rights reserved.

Bioaccumulation of the hepatotoxic microcystins in various organs of a freshwater snail from a subtropical Chinese lake, Taihu lake, with dense toxic microcystis blooms

In this paper, we describe the seasonal dynamics of three common microcystins (MCs MC-RR, MC-YR, and MC-LR) in the whole body, hepatopancreas, intestine, gonad, foot, remaining tissue, and offspring of a freshwater snail, Bellamya aeruginosa, from Gonghu Bay of Lake Taihu, China, where dense toxic Microcystis blooms occur in the warm seasons. Microcystins were determined by liquid chromatography electrospray ionization mass spectrum. Microcystin (MC-RR + MC-YR + MC-LR) content of the offspring and gonad showed high positive correlation, indicating that microcystins could transfer from adult females to their young with physiological connection. This study is the first to report the presence of microcystins in the offspring of the adult snail. The majority of the toxins were present in the intestine (53.6%) and hepatopancreas (29.9%), whereas other tissues contained only 16.5%. If intestines are excluded, up to 64.3% of the toxin burden was allocated in the hepatopancreas. The microcystin content in the intestine, hepatopancreas, and gonad were correlated with the biomass of Microcystis and intracellular and extracellular toxins. Of the analyzed foot samples, 18.2% were above the tolerable daily microcystin intake recommended by the World Health Organization (WHO) for human consumption. This result indicates that public health warnings regarding human ingestion of snails from Taihu Lake are warranted. In addition, further studies are needed to evaluate the occurrence by Microcystis in relation to spatial and temporal changes in water quality.

Relationships between microcystins and environmental parameters in 30 subtropical shallow lakes along the Yangtze River, China

1. A survey of 30 subtropical shallow lakes in the middle and lower reaches of the Yangtze River area in China was conducted during July-September in 2003-2004 to study how environmental and biological variables were associated with the concentration of the cyanobacterial toxin microcystin (MC). 2. Mean MC concentration in seasonally river-connected lakes (SL) was nearly 33 times that in permanently river-connected lakes (RL), and more than six times that in city lakes (NC) and non-urban lakes (NE) which were not connected to the Yangtze River. The highest MC (8.574 mu g L-1) was detected in Dianshan Lake. 3. MC-RR and MC-LR were the primary toxin variants in our data. MC-RR, MC-YR and MC-LR were significantly correlated with Ch1 a, biomass of cyanobacteria, Microcystis and Anabaena, indicating that microcystins were mainly produced by Microcystis and Anabaena sp. in these lakes. 4. Nonlinear interval maxima regression indicated that the relationships of Secchi depth, total nitrogen (TN) : total phosphorus UP) and NH4+ with MC were characterised by negative exponential curves. The relationships between MC and TN, TP, NO3- + NO2- were fitted well with a unimodal curve. 5. Multivariate analyses by principal component and classifying analysis indicated that MC was mainly affected by Microcystis among the biological factors, and was closely related with temperature among physicochernical factors.

In situ studies on the bioaccumulation of microcystins in the phytoplanktivorous silver carp (Hypophthalmichthys molitrix) stocked in Lake Taihu with dense toxic Microcystis blooms

The phytoplanktivorous silver carp is an important biomanipulation fish to control cyanobacterial blooms and is also a food fish with the greatest production in China. The accumulation of the hepatotoxic microcystins (MCs) determined by LC-MS in various organs of silver carp was studied monthly in Lake Taihu dominated by toxic Microcystis aeruginosa. Average recoveries of spiked fish samples were 78% for MC-RR and 81% for MC-LR. The highest content of MCs was found in the intestine (97.48 mu g g(-1) DW), followed by liver (6.84 mu g g(-1) DW), kidney (4.8 8 mu g g(-1) DW) and blood (1.54 mu g g(-1) DW), and the annual mean MC content was in the order of intestine > liver > kidney > blood > muscle > spleen > gallbladder > gill. Silver carp could effectively ingest toxic Microcystis cells (up to 84.4% of total phytoplankton in gut contents), but showed fast growth (from 141 g to 1759 g in I year in mean weight). Silver carp accumulated less microcystins in liver than other animals in the same site or other fish from different water bodies at similar level of toxin ingestion. There was possible inhibition of the transportation of the most toxic MC-LR across the gutwall. Muscle of silver carp in Lake Taihu should not be consumed during period of dense Microcystis blooms while viscera were risky for consumption in more months. (c) 2006 Elsevier B.V. All rights reserved.

The impact of planktivorous fishes on microcystin concentrations in Meiliang Bay, Lake Taihu, China

In an eight-month enclosure experiment in Meiliang Bay of Lake Taihu, a shallow subtropical lake in China, silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) collectively reduced cyanobacterial biomass. Microcystin concentration was six times higher in the 0.35 km(2) control enclosure (without fish) than in two similar-sized enclosures that had been stocked with both carp species. Furthermore, toxic Microcystis spp. increased microcystin production when exposed to silver carp and bighead carp.

Sorption, degradation and mobility of microcystins in Chinese agriculture soils: Risk assessment for groundwater protection

In the present paper, sorption, persistence, and leaching behavior of three microcystin variants in Chinese agriculture soils were examined. Based on this study, the values of capacity factor and slope for three MCs variants in three soils ranged from 0.69 to 6.00, and 1.01 to 1.54, respectively. The adsorption of MCs in the soils decreased in the following order: RR > Dha(7) LR > LR. Furthermore, for each MC variant in the three soils, the adsorption rate in the soils decreased in the following order: soil A > soil C > soil B. The calculated half-time ranged between 7.9 and 17.8 days for MC-RR, 6.0-17.1 days for MC-LR, and 7.1-10.2 days for MC-Dha(7) LR. Results from leaching experiments demonstrated that recoveries of toxins in leachates ranged from 0-16.7% for RR, 73.2-88.9% for LR, and 8.9-73.1% for Dha 7 LR. The GUS value ranged from 1.48 to 2.06 for RR, 1.82-2.88 for LR, and 1.76-2.09 for Dha(7) LR. Results demonstrated the use of cyanobacterial collections as plant fertilizer is likely to be unsafe in soils. (c) 2006 Elsevier Ltd. All rights reserved.

Microcystin-RR-induced apoptosis in tobacco BY-2 cells

Our previous studies showed that microcystin-RR could induce oxidative damage in plant cells as they do with animal cells. However, whether microcystin can induce plant cell apoptosis is still unknown. In this study, the morphological changes of tobacco BY-2 suspension cells exposed to microcystin-RR were observed under light microscopy and transmission electron microscopy, and apoptosis was clearly distinguished by intense perinuclear chromatin margination, condensation of nuclear chromatin after 6d exposure of 50 mg/L (about 50 mu M) microcystin-RR. We also found that microcystin-RR can induce tobacco cell apoptosis in a dose- and time-dependent manner with flow cytometry analysis. Our study provides the first evidence that microcystins can induce plant cell apoptosis. (c) 2006 Elsevier Ltd. All rights reserved.

Optimization of an effective extraction procedure for the analysis of microcystins in soils and lake sediments

Microcystin analysis in sediments and soils is considered very difficult due to low recovery for extraction. This is the primary limiting factor for understanding the fate of toxins in the interface between water and sediment in both the aquatic ecosystem as well as in soils. In the present study, a wide range of extraction solvents were evaluated over a wide range of pH, extraction approaches and equilibration time to optimize an effective extraction procedure for the analysis of microcystins in soils and lake sediments. The number of extractions required and acids in extraction solutions were also studied. In this procedure, EDTA-sodium pyrophosphate solution was selected as an extraction solvent based on the adsorption mechanism study. The optimized procedure proved to be highly efficient and achieved over 90% recovery. Finally, the developed procedure was applied to field soil and sediment sample collected from Chinese lakes during bloom seasons and microcystins were determined in six of ten samples. (c) 2005 Elsevier Ltd. All rights reserved.

Responses of antioxidant system in Arabidopsis thaliana suspension cells to the toxicity of microcystin-RR

Microcystins are cyclic heptapeptide hepatoxins produced by many species of cyanobacteria. The toxic effects and mechanism of microcystins on animals have been well studied both in vivo and in vitro. It was also reported that microcystins had adverse effects on plants. However, to our knowledge, there is no information about the toxic effects and mechanism of microcystins on plant suspension cells. In this study, Arabidopsis thaliana suspension cells were exposed to a range dose of microcystin-RR. Lipid peroxidation, a main manifestation of oxidative damage, was studied and a time- and dose-dependent increase in malondiadehyde was observed. In contrast, glutathione (GSH) levels in the cells decreased after 48 h treatment with 1 and 5 mg/L of microcystin-RR. The activities of superoxide dismutase (SOD) and catalase (CAT) increased significantly after 48 h exposure to I and 5 mg/L of microcystin-RR, but glutathione S-transferase (GST) activity showed no difference compared with the control. These results clearly indicate that microcystin-RR is able to cause oxidative damage in A. thaliana suspension cells. Decrease of GSH content and increases of SOD and CAT activities reveal that the antioxidant system may play an important role in eliminating or alleviating the toxicity of microcystin-RR. The possible toxicity mechanism of microcystin-RR on the A. thaliana suspension cells is also discussed in this paper. (C) 2005 Elsevier Ltd. All rights reserved.