Mechanism study on the frequent variations of cell-bound microcystins in cyanobacterial blooms in Lake Taihu: Implications for water quality monitoring and assessments

Although Microcystis-based toxins have been intensively studied, previous studies using laboratory cultures of Microcystis strains are difficult to explain the phenomenon that microcystin concentrations and toxin variants in natural blooms differ widely and frequently within a short-term period. The present study was designed to unravel the mechanisms for the frequent variations of intracellular toxins related to the differences in cyanobacterial colonies during bloom seasons in Lake Taihu, China. Monitoring of Microcystis colonies during warm seasons indicated that the variations in microcystins in both concentrations and toxin species were associated with the frequent alteration of Microcystis colonies in Lake Taihu. High concentration of microcystins in the blooms was always associated with two Microcystis colonies, Microcystis flos-aquae and Microcystis aeruginosa, whereas when Microcystis wesenbergii was the dominant colonial type, the toxin production of the blooms was low. Additionally, environmental factors such as temperature and nutrition were also shown to have an effect on the toxin production of the blooms, and may also potentially influence the Microcystis species present. The results of the present study provides insight into a new consideration for quick water quality monitoring, assessment and risk alert in cyanobacterium- and toxin-contaminated freshwaters, which will be beneficial not only for water agencies but also for public health. (C) 2009 Elsevier Ltd. All rights reserved.

Detection of microcystins in environmental samples using surface plasmon resonance biosensor

An indirect inhibitive surface plasmon resonance (SPR) immunoassay was developed for the microcystins (MCs) detection. The bioconjugate of MC-LR and bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. A serial premixture of MC-LR standards (or samples) and monoclonal antibody (mAb) were injected over the functional sensor surface, and the subsequent specific immunoreaction was monitored on the BIAcore 3000 biosensor and generated a signal with an increasing intensity in response to the decreasing MCs concentration. The developed SPR immunoassay has a wide quantitative range in 1-100 mu g L-1. Although not as sensitive as conventional enzyme-linked immunosorbent assay (ELISA), the SPR biosensor offered unique advantages: (I) the sensor chip could be reusable without any significant loss in its binding activity after 50 assay-regeneration cycles, (2) one single assay could be accomplished in 50 min (including 30-min preincubation and 20-min BIAcore analysis), and (3) this method did not require multiple steps. The SPR biosensor was also used to detect MCs in environmental samples, and the results compared well with those obtained by ELISA. We conclude that the SPR biosensor offers outstanding advantages for the MCs detection and may be further developed as a field-portable sensor for real-time monitoring of MCs on site in the near future. (C) 2009 Published by Elsevier B.V.

Rapid quantification of total microcystins in cyanobacterial samples by periodate-permanganate oxidation and reversed-phase liquid chromatography

Microcystins (MCs) comprise a family of more than 80 related cyclic hepatotoxic heptapeptides. Oxidation of MCs causes cleavage of the chemically unique C-20 beta-amino acid (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) amino to form 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB), which has been exploited to enable analysis of the entire family. In the present study, the reaction conditions (e.g. concentration of the reactants. temperature and pH) used in the production of MMPB by oxidation of cyanobacterial samples with permanganate-periodate were optimized through a series of well-controlled batch experiments. The oxidation product (MMPB) was then directly analyzed by high-performance liquid chromatography with diode array detection. The results of this study provided insight into the influence of reaction conditions on the yield of MMPB. Specifically, the optimal conditions, including a high dose of permanganate (>= 50 mM) in saturated periodate solution at ambient temperature under alkaline conditions (pH similar to 9) over 1-4 h were proposed, as indicated by a MMPB yield of greater than 85%. The technique developed here was applied to determine the total concentration of MCs in cyanobacterial bloom samples, and indicated that the MMPB technique was a highly sensitive and accurate method of quantifying total MCs. Additionally, these results will aid in development of a highly effective analytical method for detection of MMPB as an oxidation product for evaluation of total MCs in a wide range of environmental sample matrices, including natural waters, soils (sediments) and animal tissues. (C) 2009 Elsevier B.V. All rights reserved.

Hepatic Histopathological Characteristics and Antioxidant Response of Phytoplanktivorous Silver Carp Intraperitoneally Injected with Extracted Microcystins

Objective To investigate the hispathological characteristics and antioxidant responses in liver of silver carp after intraperitoneal administration of microcystins (MCs) for further understanding hepatic intoxication and antioxidation mechanism in fish. Methods Phytoplanktivorous silver carp was injected intraperitoneally (i.p.) with extracted hepatotoxic microcystins (mainly MC-RR and -LR) at a dose of 1000 mu g MC-LReq./kg body weight, and liver histopathological changes and antioxidant responses were studied at 1, 3, 12, 24, and 48 h, respectively, after injection. Results The damage to liver structure and the activities of hepatic antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxide (GPX) were increased in a time-dependent manner. Conclusion In terms of clinical and histological signs of intoxication and LD50 (i.p.) dose of MC-LR, silver carp appears rather resistant to MCs exposure than other fishes. Also, the significantly increased SOD activity in the liver of silver carp suggests a higher degree of response to MCs exposure than CAT and GPX.

Involvement of Fas/FasL system in apoptotic signaling in testicular germ cells of male Wistar rats injected i.v. with microcystins

Previous studies have shown that gonads were the second target organ of microcystins (MCs), and that MCs exposure exerted obvious toxic effects on male reproductive system of mammals. However, relevant molecular evidences are still lacking. Fas-signaling pathway plays a key role in toxicant-induced germ cell apoptosis. This study was to evaluate the responses of Fas/FasL system related genes and proteins in testes of rats injected intravenously with MCs. Enhanced apoptosis of germ cells in the testes of MCs-treated rats was detected by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) associated with up-regulation of the Fas/FasL system. Both Fas and FasL protein expression were induced evidently from I h post-injection, and this high expression level maintained throughout the experiment. In addition, the activation of caspase-8 and caspase-3 protein was also observed, which were indicators of apoptosis. These results suggested the likely involvement of Fas/FasL system in the MCs-induced germ cell apoptosis. It is also suggested that MCs can cause damage to Sertoli cells directly. (C) 2009 Elsevier Ltd. All rights reserved.

Intraperitoneal injection of extracted microcystins results in hypovolemia and hypotension in crucian carp (Carassius auratus)

Circulatory responses of crucian carp injected intraperitoneally with extracted micro-cystins (MCs) were studied at sublethal and lethal doses (150 and 600 mu g MC kg(-1) body mass, respectively). Mean arterial blood pressure (MAP), heart rate, hematocrit (Hct), red blood cell (RBC) counts, and circulating blood volume (BV) were assayed at 0, 1, 3, 12, 24, and 48 h post-toxin administration. MAP decreased significantly in a dose-dependent manner over time. Within the 48-h test period, the lethal dose as well as the sublethal dose resulted in a steady decline of MAP without recovery. Heart rate significantly increased within 24 h post-injection as blood pressure significantly dropped, then showed a terminal decline to the control level. The dose-dependent decreases in BV and Hct were directly related to the drop in MAP. Intraperitoneal injection of a lethal dose of MCs led to hepatic and gill hemorrhage. Consequently, crucian carp given MCs suffered from hypovolemic hypotensive shock. (C) 2009 Elsevier Ltd. All rights reserved.

Transfer, distribution and bioaccumulation of microcystins in the aquatic food web in Lake Taihu, China, with potential risks to human health

In this paper, accumulation and distribution of microcystins (MCs) was examined monthly in six species of fish with different trophic levels in Meiliang Bay, Lake Taihu, China, from June to November 2005, Microcystins were analyzed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). Average recoveries of spiked fish samples were 67.7% for MC-RR, 85.3% for MC-YR, and 88.6% for MC-LR. The MCs (MC-RR+MC-YR+MC-LR) concentration in liver and gut content was highest in phytoplanktivorous fish, followed by omnivorous fish, and was lowest in carnivorous fish; while MCs concentration in muscle was highest in omnivorous fish, followed by phytoplanktivorous fish, and was lowest in carnivorous fish. This is the first study reporting MCs accumulation in the gonad of fish in field. The main uptake of MC-YR in fish seems to be through the gills from the dissolved MCs. The WHO limit for tolerable daily intake was exceeded only in common carp muscle. (C) 2008 Elsevier B.V. All rights reserved.

Oxidative stress response after prolonged exposure of domestic rabbit to a lower dosage of extracted microcystins

Oxidative stress response after prolonged exposure to a low dose of microcystins (MCs) was studied in liver, kidney and brain of domestic rabbits. Rabbits were treated with extracted MCs (mainly MC-LR and MC-RR) at a dose of 2 MC-LReq. mu g/kg body weight or saline solution every 24 h for 7 or 14 days. During the exposure of MCs, increase of lipid peroxidation (LPO) levels were detected in all the organs studied, while antioxidant enzymes responded differently among different organs. The enzyme activities Of Superoxide dismutase (SOD). catalase (CAT) and glutathione reductase (GR) in liver decreased in the MCs treated animals. In brain, there were obvious changes in glutathione peroxidase (GPx) and GR, while only CAT was obviously influenced in kidney. Therefore, daily exposure at a lower dosage of MCs, which mimicked a natural route of MCs. could also induce obvious oxidative stress in diverse organs of domestic rabbits. The oxidative stress induced by MCs in brain was as serious as in liver and kidney, suggesting that brain may also be a target of MCs in mammals. And it seems that animals may have more time to metabolize the toxins or to form an adaptive response to reduce the adverse effects when exposed to the low dose of MCs. (C) 2008 Elsevier B.V. All rights reserved.

Time-Dependent Oxidative Stress Responses of Crucian Carp (Carassius auratus) to Intraperitoneal Injection of Extracted Microcystins

This study was conducted to investigate time-dependent changes in oxidative enzymes in liver of crucian carp after intraperitoneally injection with extracted microcystins 600 and 150 mu g kg(-1) body weight. The results showed that activities of antioxidant enzymes, including superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase generally exhibited a rapid increase in early phase (1-3 h post injection), but gradually decreased afterwards (12-48 h) compared with the control, with an evident time-dependent effect. These zigzag changes over time contributed a better understanding on oxidative stress caused by microcystins in fish.

First Identification of the Hepatotoxic Microcystins in the Serum of a Chronically Exposed Human Population Together with Indication of Hepatocellular Damage

Hepatotoxic microcystins (MCs) are the most commonly reported cyanotoxins in eutrophic freshwaters. In 1996, human intoxications by MCs caused deaths of 76 patients at Caruaru dialysis centers in Brazil. So far, there have been no direct evidences of MC occurrence in human tissue in consequence of exposure to MC. In this study, we improved cleanup procedures for detecting MCs in serum sample using liquid chromatographymass spectrometry, and confirmed for the first time the presence of MCs in serum samples (average 0.39 ng/ml, which amounts to ca. 1/87 of the concentrations found in tissue samples of the Caruaru victims) of fishermen at Lake Chaohu. Daily intake by the fishermen was estimated to be in the range of 2.2-3.9 mu g MC-LReq, whereas the provisional World Health Organization tolerable daily intake (TDI) for daily lifetime exposure is 0.04 mu g/kg or 2-3 mu g per person. Moreover, statistical analysis showed closer positive relationships between MC serum concentrations and concentrations of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase than between the MC concentrations and other biochemical indicators. Thus, the data raise the question whether extended exposure in the range of the TDI or up to a factor of 10 above it may already lead to indication of liver damage. The results also demonstrate a risk of health effects from chronic exposure to MCs at least for populations with high levels of exposure, like these fishermen.

Changes in plasma thyroid hormones and cortisol levels in crucian carp (Carassius auratus) exposed to the extracted microcystins

The endocrine response of crucian carp injected intraperitoneally with extracted microcystins (MC) was investigated in this study. Fish were injected intraperitoneally either with 0.75% NaCl (control) and Microcystis extract corresponding to 150 and 600 mu g microcystins per kg body weight. The plasma levels of triiodothyronine (T-3), thyroxine (T-4), free triiodothyronine (FT3), free thyroxine (FT4), and cortisol were determined at 0, 1, 3, 12, 24. and 48 h post-administration of MC-containing extract. Treated fish displayed abnormal behaviors, Such as a startle response and disoriented swimming, as well as changes in ventilation rates. Plasma cortisol concentrations of fish in both dose groups significantly increased after administration of extracted MC and remained high throughout the experiment, which suggested that MC elicited a stress response in treated fish. The profiles of cortisol changes in treated fish appeared to be dose dependent, indicating that fish in the high dose group experienced greater MC-incluced disturbance. Mortality occurred after 12 h in the high dose group. Plasma levels of T-4, T-3, FT4, and FT3 did not vary significantly between the control fish. In contrast to this, fish exposed to MC-containing extract showed significant declines in T-3, FT4, and FT3 levels in a dose-depenclent manner throughout the experiment. Plasma T4 levels, however, did not vary significantly in the low dose group, whereas they decreased significantly it 48 h post injection in the high dose group. This study demonstrates that administration of microcystins-containing extract causes a stress response and reduces the plasma levels of thyroid hormones in crucian carp. These results illustrate that microcystins exerted potent effects on the endocrine system of crucian carp, through activating their hypothalamus-pituitary- interrenal axis and disturbing thyroid function. (c) 2008 Elsevier Ltd. All rights reserved.

Distribution of microcystins in various organs (heart, liver, intestine, gonad, brain, kidney and lung) of Wistar rat via intravenous injection

The distribution of microcystins (MCs) in various tissues of Wistar rats was studied under laboratory conditions. Rats were injected intravenously (i.v.) with extracted MCs at a dose of 80 mu g MC-LRequivalent/kg body weight. MCs concentrations in various tissues were detected at 1, 2. 4, 6, 12 and 24 h post-injection using liquid chromatography-mass spectrometry (LC-MS). The highest concentration of MCs was found in kidney (0.034-0.295 mu g/g dry weight), followed by lung (0.007-0.067 mu g/g dry weight), stomach (0.010-0.058 mu g/g dry weight) and liver (0.003-0.052 mu g/g dry weight). The maximum MCs content in the whole body of rat, 2.9% of the injected dose, was observed at 2 h post-injection. MCs concentration was higher in kidney than in liver during the experiment, and two peaks of MCs concentration (at 2 and 24 h, respectively) were observed in kidney, indicating that MCs can be excreted directly via kidney of rat. Though heart, intestine, spleen, brain, gonad and stomach contained less than 0.2% of injected MCs during the whole experiment stage, the presence of MCs in these tissues represents potential damage to them. (c) 2008 Elsevier Ltd. All Fights reserved.

Spatial and temporal variations of microcystins in hepatopancreas of a freshwater snail from Lake Taihu

In this paper, spatial and temporal variations of three common microcystins (MC-RR, MC-YR, and MC-LR) in the hepatopancreas of a freshwater snail (Bellamya aeruginosa) were studied monthly in two bays of Lake Taihu. Microcystins (MCs) concentration in hepatopancreas was quantified by liquid chromatography-mass spectrometry (LC-MS). The MCs concentrations in hepatopancreas were higher at Site 1 than those at other sites, which was in agreement with the changes of intracellular MCs concentrations in the water column. There was a significant correlation between MCs concentrations in the hepatopancreas and that in the seston, suggesting that spatial variances of MCs; concentrations in hepatopancreas among the five sites were due to spatial changes of toxic Microcystis cells in the water column. PCCA indicates that in addition to Microcystis, other factors (e.g., water temperature) also substantially affected the accumulation of MCs in hepatopancreas of the snail. (C) 2008 Published by Elsevier Inc.

Quantitative Determination of Microcystins in Rat Plasma by LC-ESI Tandem MS

A rapid and sensitive method was developed and validated for the determination of MCYST (microcystin)-RR, -LR, and [Dha(7)] MCYST-LR in rat plasma by liquid chromatography-tandem mass spectrometry. The analytes were extracted from rat plasma by protein precipitation, followed by solid-phase extraction. Liquid chromatography with electrospray ionization mass spectrometry, operating in selected reaction monitoring (SRM) mode, was used to quantify MCYST-RR, -LR, and [Dha(7)] MCYST-LR in rat plasma. The recoveries for each analyte in rat plasma ranged from 70.8 to 88.7%. The calibration curve was linear within the range from 0.005 to 1.25 mu g mL(-1). The limit of detection were 1.4, 1.0, 0.6 ng mL(-1) for MCYST-RR, -LR, and [Dha(7)] MCYST-LR. The overall precision was determined on three different days. The values for within- and between-day precision in rat plasma were within 15%. This method was applied to the identification and quantification of microcystins in rat plasma with acute exposure of microcystins via intravenous injection.