Determinations of MC-LR and [Dha(7)] MC-LR Concentrations and Physicochemical Properties by Liquid Chromatography-Tandem Mass Spectrometry

A liquid chromatography electrospray mass spectrometry (LC/ESI/MS) method working in multiple reactions monitoring mode for the determination of trace amounts of microcystin variants (MC-LR and [Dha(7)] MC-LR) in waters was developed. The limit of quantification was 0.05 mu g/L and the limit of detection was 0.015 mu g/L for MC-LR and [Dha(7)] MC-LR, respectively. Recoveries for MCs were in the range of 68%-81%. MC-LR and [Dha(7)] MC-LR were chemically stable with similar physiochemical behavior.

微囊藻毒素[Dha~7]MCRR的制备及鉴定

以野外收集的水华蓝藻为原料,经过75%甲醇溶液浸提,快速色谱分离和半制备色谱纯化,从滇池水华蓝藻中分离纯化出1种微囊藻毒素变体.电喷雾质谱、紫外分光光度计和HPLC检测结果表明,所得毒素为[Dha7]MCRR,是MCRR的1种去甲基化变体,其纯度大于95%.该毒素的分子组成为环(Ala-Arg-MeAsp-Arg-Adda-Glu-Dha),分子量为1 023,其紫外扫描光谱(200~300 nm)在239 nm处有特征吸收.[Dha7]MCRR在滇池水华蓝藻中普遍存在,有时会成为MC的主要种类.

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.

微囊藻毒素LR诱导无DNA片断化的细胞凋亡

目的研究微囊藻毒素(microcystin,MC)损伤肝细胞的毒性机制。方法以不同浓度的微囊藻毒素LR(MCLR)处理L-02肝细胞,通过光镜和电镜下的形态观察、DNA片断化分析、线粒体膜电位变化等了解MCLR对肝细胞的毒性效应。结果光镜下观察表明,50!g/mlMCLR处理可使细胞变圆、萎缩、不贴壁生长,电镜下L-02细胞呈现皱缩、膜发泡及染色质凝聚并边缘化等变化,这是典型的细胞凋亡形态特征,但电镜下没有观察到凋亡小体的形成;DNA电泳表明凋亡细胞没有出现明显的DNAladder,与电镜观察的结果一致。

Determination of microcystin-LR and its metabolites in snail (Bellamya aeruginosa), shrimp (Macrobrachium nipponensis) and silver carp (Hypophthalmichthys molitrix) from Lake Taihu, China

This paper describes seasonal changes of microcystin-LR (MC-LR) and its glutathione (MC-LR-GSH) and cysteine conjugates (MC-LR-Cys) in three aquatic animals - snail (Bellamya aeruginosa), shrimp (fMacrobrachium nipponensis) and silver carp (Hypophthalmichthys molitrix) collected from Lake Taihu, China. MC-LR, MC-LR-GSH, and MC-LR-Cys were determined by liquid chromatography electrospray ionization mass spectrum (LC-ESI-MS). The mean MC-LR concentrations in the hepatopancreas of snail and shrimp and liver of silver carp were 6.61, 0.24, and 0.027 mu g g(-1) dry weight (DW), respectively: while the average MC-LR-Cys concentrations were 0.50, 0.97, and 5.72 mu g g(-1) DW, respectively. MC-LR-GSH was usually not detectable in these samples. The above results suggest that: (1) in aquatic animals, especially fish, the main excretion form of MC-LR could be MC-LR-Cys, but not MC-LR-GSH, whereas MC-LR-Cys might play an important role in detoxication of MC-LR and (2) that efficiency of MC-LR-Cys formation differs among species. The main detoxication pathway of MC-LR in aquatic animals is suggested as follows: when MC-LR enters into liver/hepatopancreas, it firstly conjugates with polypeptide or protein (including GSH, PP-1 and 2A) containing Cys residues, perhaps also some free cysteine; subsequently, MC-LR-Cys is degraded from these polypeptide or protein; and finally is excreted from animals by the compound of MC-LR-Cys. (C) 2009 Elsevier Ltd. All rights reserved.

Ultrastructural alteration of lymphocytes in spleen and pronephros of grass carp (Ctenopharyngodon idella) experimentally exposed to microcystin-LR

Relatively little is known in relation to pathological changes of immune organs in fish when exposed to MC-LR. The ultrastructural alteration of lymphocytes was examined in the spleen and pronephros of grass carp Ctenopharyngodon idella injected experimentally with microcystin-LR. The fish were intraperitoneally injected with MC-LR at a dose of 50 mu g/kg body weight, and the spleen and pronephros were dissected out at 1, 2, 7, 14 and 21 days post intraperitoneal injection (dpi). Pathological changes were then examined by transmission electron microscopy. Apoptosis was detected only in lymphocytes in the spleen, with obvious apoptotic features observed at 2 dpi; pathological changes of lymphocytes in the pronephros were also serious with mitochondria being highly edematous. However, damaged lymphocytes were almost un-observed in the spleen and pronephros at 21 dpi. These findings suggest that MC-LR can induce toxic effect on immune organs in grass carp, and the spleen may be much more sensitive to MC-LR stimulation. (C) 2008 Elsevier B.V. All rights reserved.

Simultaneous determination of microcystin-LR and its glutathione conjugate in fish tissues by liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantitative determination of microcystin-LR (MC-LR) and its glutathione conjugate (MC-LR-GSH) in fish tissues. The analytes were extracted from fish liver and kidney using 0.01 M EDTA-Na-2-5% acetic acid, followed by a solid-phase extraction (SPE) on Oasis HLB and silica cartridges. High-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry, operating in selected reaction monitoring (SRM) mode, was used to quantify MC-LR and its glutathione conjugate in fish liver and kidney. Recoveries of analytes were assessed at three concentrations (0.2, 1.0, and 5 mu g g(-1) dry weight [DW]) and ranged from 91 to 103% for MC-LR, and from 65.0 to 75.7% for MC-LR-GSH. The assay was linear within the range from 0.02 to 5.0 mu g g(-1) DW, with a limit of quantification (LOQ) of 0.02 mu g g(-1) DW. The limit of detection (LOD) of the method was 0.007 mu g g(-1) DW in both fish liver and kidney. The overall precision was determined on three different days. The values for within- and between-day precision in liver and kidney were within 15%. This method was applied to the identification and quantification of MC-LR and its glutathione conjugate in liver and kidney of fish with acute exposure of MC-LR. (c) 2007 Elsevier B.V. All rights reserved.

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.

Tissue distributions and seasonal dynamics of the hepatotoxic microcystins-LR and -RR in a freshwater snail (Bellamya aeruginosa) from a large shallow, eutrophic lake of the subtropical China

Tissue distributions and seasonal dynamics of the hepatotoxic microcystins-LR and -RR in a freshwater snail (Bellamya aeruginosa) were studied monthly in a large shallow, eutrophic lake of the subtropical China during June-November, 2003. Microcystins (MCs) were quantitatively determined by High-Performance Liquid Chromatography (HPLC) with a qualitative analysis by a Finnigan LC-MS system. On the average of the study period, hepatopancreas was the highest in MC contents (mean 4.14 and range 1.06-7.42 mug g(-1) DW), followed by digestive tracts (mean 1.69 and range 0.8-4.54 mug g(-1) DW) and gonad (mean 0.715 and range 0-2.62 mug g(-1) DW), whereas foot was the least (mean 0.01 and range 0-0.06 mug g(-1) DW). There was a positive correlation in MC contents between digestive tracts and hepatopancreas. A constantly higher MC content in hepatopancreas than in digestive tracts indicates a substantial bioaccumulation of MCs in the hepatopancreas of the snail. The average ratio of MC-LR/MC-RR showed a steady increase from digestive tracts (0.44) to hepatopancreas (0.63) and to gonad (0.96), suggesting that MC-LR was more resistant to degradation in the snail. Since most MCs were present in the hepatopancreas, digestive tracts and gonad with only a very small amount in the edible foot, the risk to human health may not be significant if these toxic parts are removed prior to snail consumption. However, the possible transference of toxins along food chains should not be a negligible concern. (C) 2004 Elsevier Ltd. All rights reserved.

Dynamics of microcystins-LR and -RR in the phytoplanktivorous silver carp in a sub-chronic toxicity experiment

A sub-chronic toxicity experiment was conducted to examine tissue distribution and depuration of two microcystins (microcystin-LR and microcystin -RR) in the phytoplanktivorous filter-feeding silver carp during a course of 80 days. Two large tanks (A, B) were used, and in Tank A, the fish were fed naturally with fresh Microcystis viridis cells (collected from a eutrophic pond) throughout the experiment, while in Tank B, the food of the fish were M. viridis cells for the first 40 days and then changed to artificial carp feed. High Performance Liquid Chromatography (HPLC) was used to measure MC-LR and MC-RR in the M. viridis cells, the seston, and the intestine, blood, liver and muscle tissue of silver carp at an interval of 20 days. MC-RR and MC-LR in the collected Microcystis cells varied between 268-580 and 110-292 mug g(-1) DW, respectively. In Tank A, MC-RR and MC-LR varied between 41.5-99.5 and 6.9-15.8 mug g(-1) DW in the seston, respectively. The maximum MC-RR in the blood, liver and muscle of the fish was 49.7, 17.8 and 1.77 mug g(-1) DW, respectively. No MC-LR was detectable in the muscle and blood samples of the silver carp in spite of the abundant presence of this toxin in the intestines (for the liver, there was only one case when a relatively minor quantity was detected). These findings contrast with previous experimental results on rainbow trout. Perhaps silver carp has a mechanism to degrade MC-LR actively and to inhibit MC-LR transportation across the intestines. The depuration of MC-RR concentrations occurred slowly than uptakes in blood, liver and muscle, and the depuration rate was in the order of blood > liver > muscle. The grazing ability of silver carp on toxic cyanobacteria suggests an applicability of using phytoplanktivorous fish to counteract cyanotoxin contamination in eutrophic waters. (C) 2003 Elsevier Ltd. All rights reserved.

The toxic effects of microcystin-LR on embryo-larval and juvenile development of loach, Misguruns mizolepis Gunthe

Microcystin-LR, a specific and potent hepatotoxin, was tested for its effects oil loach embryo-larval and juvenile development, The results of this study showed that loach embryos were more sensitive when exposed to microcystin-LR at a later than at an earlier stage of development, Juveniles were far less sensitive to MC-LR than were embryos and larvae. Mortality and developmental abnormality were proven to be dose-dependent and to be stage-specific sensitive. Among the abnormal changes noted were: pericardial edema and tubular heart, bradycardia, homeostasis, poor yolk resumption. small head, curved body and tail, and abnormal hatching, Liver and heart were the main targets of microcystin-LR toxicity. Ultrastructural analysis documented a complex set of sublethal effects of microcystin-LR on loach hepatocytes, chiefly including morphological alteration in nuclear and RER of loach liver cells. fit addition, microcystin-LR was lethal to loach juvenile in the subacute (7 days) exposure (LC50) = 593.3 mug/l). (C) 2002 Elsevier Science Ltd. All rights reserved.

Efeitos do extrato bruto e da microcistina-LR em Lactuca sativa L. (Asteraceae)

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

微囊藻毒素LR诱导L-02细胞发生凋亡

目的了解微囊藻毒素(MC)LR对L-02细胞的毒性机制。方法以L-02细胞为材料,用不同浓度的MCLR处理该细胞,观察了细胞增殖能力、细胞形态改变、乳酸脱氢酶(LDH)泄漏、细胞凋亡率及凋亡相关基因等一系列指标的变化。结果 MTT细胞增殖实验可知,MCLR在24 h内对L-02细胞有轻微的抑制作用,随后却促进细胞增殖。48 h处理对 LDH泄漏没有显著影响,延时处理导致LDH泄漏发生,且MCLR浓度越高,LDH泄漏越严重,此结果显示发生了细胞氧化损伤。光镜下50μg/ml的毒素浓度在60 h处理后可造成明

天然水体中痕量微囊藻毒素的高效液相色谱测定方法优化

以微囊藻毒素LR(MC-LR)和微囊藻毒素RR(MC-RR)为材料,主要通过调节杂质淋洗液和毒素洗脱液中甲醇、水和三氟乙酸(TFA)的配比来改变极性和PH值,对高效液相色谱(HPLC)法分析MC环境样品的方法进行了优化。结果表明,含0.1%TFA的40%~45%的甲醇水溶液可以取得较好的杂质淋洗效果;含0.1%TFA的70%的甲醇水溶液可以将固相萃取柱(SPE)上的MC完全洗下。因此,建议在分析杂质较多的环境样品时,使用含0.1%TFA的40%~45%的甲醇水溶液对杂质进行淋洗,然后用含0.1%TFA的7

Effects of cyanobacterial toxin microcystin-LR on the transcription levels of immune-related genes in grass carp Ctenopharyngodon idella

Recent studies in mammals have revealed that the cyanobacterial toxin MC-LR suppresses immune functions. Nevertheless, immunotoxic effects of microcystins have been little studied in fish. In this paper, we present the profiles of the immune modulation of MC-LR in grass carp, and quantitative real-time PCR methodology was developed for the measurement of relative transcription changes of six immune-related genes in the spleen and head kidney of the grass carp Ctenopharyngodon idella, which were intraperitoneally injected with 50 mu g MC-LR center dot kg(-1) body weight in a three-week period. This study was focused exclusively on gene transcription level changes at different time points after MC-LR exposure, so, only one dose was given. The investigated genes were interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), type I interferon (Type I IFN), peptidoglycan recognition protein-L (PGRP-L), immunoglobulin M (IgM) and major histocompatibility complex class I (MHC-I) genes. The results demonstrated that the transcription levels of the TNF-alpha, type I IFN, and PGRP-L genes in the spleen and head kidney were significantly low at all time points, and those of IL-1 beta were significantly low in the head kidney at different time points. In addition, IgM and MHC-I transcription levels were only significantly low in the spleen and head kidney at 21 d postinjection. The changes in the transcription levels of immune-related genes induced by MC-LR confirmed its effect on inhibiting immune function at the transcription level.