Sensitivity to microcystins: a comparative study in human cell lines with and without multidrug resistance phenotype

Multidrug resistance (MDR) is an obstacle in cancer treatment. An understanding of how tumoral cells react to oxidants can help us elucidate the cellular mechanism involved in resistance. Microcystins are cyanobacteria hepatotoxins known to generate oxidative stress. The aim of this study was to compare the sensitivity to microcystins of human tumoral cell lines with (Lucena) and without (K562) MDR phenotype. Endpoints analyzed were effective microcystins concentration to 50% of exposed cells (EC50), antioxidant enzyme activity, lipid peroxidation, DNA damage, reactive oxygen species (ROS) concentration, and tubulin content. Lucena were more resistant and showed lower DNA damage than K562 cells (P<0.05). Although microcystins did not alter catalase activity, a higher mean value was observed in Lucena than in K562 cells. Lucena cells also showed lower ROS concentration and higher tubulin content. The higher metabolism associated with the MDR phenotype should increase ROS concentration and make for an improved antioxidant defense against the toxic effects of microcystins.     

Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

Background  

Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1) and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis.     

微囊藻毒素生物治理技术研究进展*

近年来,随着全球气候变暖和水体富营养化程度加深,蓝藻水华频繁暴发。微囊藻毒素是有害蓝藻产生及释放的危害最大的一类蓝藻毒素,对生态环境和公众健康造成了严重的威胁。因此,寻求有效的微囊藻毒素降解方法已成为全球科学领域的研究热点。针对微囊藻毒素生物治理技术展开综述,阐述了微囊藻毒素的产生、理化性质及生物毒性,总结了微生物、水生植物、浮游动物等自然生物降解微囊藻毒素的能力。在此基础上概述了生物滤池、人工湿地、生态浮床、膜生物膜反应器等生物治理技术对微囊藻毒素的去除效果,分析了现有微囊藻毒素生物处理方法的优势和局限性,并对今后的研究方向提出展望,为解决水环境中微囊藻毒素的污染问题提供思路。     

Distribution of Microcystins in a Lake Foodweb: No Evidence for Biomagnification

Microcystins, toxins produced by cyanobacteria, may play a role in fish kills, although their specific contribution remains unclear. A better understanding of the eco-toxicological effects of microcystins is hampered by a lack of analyses at different trophic levels in lake foodwebs. We present 3 years of monitoring data, and directly compare the transfer of microcystin in the foodweb starting with the uptake of (toxic) cyanobacteria by two different filter feeders: the cladoceran Daphnia galeata and the zebra mussel Dreissena polymorpha. Furthermore foodwebs are compared in years in which the colonial cyanobacterium Microcystis aeruginosa or the filamentous cyanobacterium Planktothrix agardhii dominated; there are implications in terms of the types and amount of microcystins produced and in the ingestion of cyanobacteria. Microcystin concentrations in the seston commonly reached levels where harmful effects on zooplankton are to be expected. Likewise, concentrations in zooplankton reached levels where intoxication of fish is likely. The food chain starting with Dreissena (consumed by roach and diving ducks) remained relatively free from microcystins. Liver damage, typical for exposure to microcystins, was observed in a large fraction of the populations of different fish species, although no relation with the amount of microcystin could be established. Microcystin levels were especially high in the livers of planktivorous fish, mainly smelt. This puts piscivorous birds at risk. We found no evidence for biomagnification of microcystins. Concentrations in filter feeders were always much below those in the seston, and yet vectorial transport to higher trophic levels took place. Concentrations of microcystin in smelt liver exceeded those in the diet of these fish, but it is incorrect to compare levels in a selected organ to those in a whole organism (zooplankton). The discussion focuses on the implications of detoxication and covalent binding of microcystin for the transfer of the toxin in the foodweb. It seems likely that microcystins are one, but not the sole, factor involved in fish kills during blooms of cyanobacteria.     

Oxidative stress response of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. (PCC 6803) due to exposure to microcystin-LR and cell-free cyanobacterial crude extract containing microcystin-LR

Cyanobacteria are able to produce a variety of secondary metabolites such as the microcystins. The ecological role of microcystins for the cyanobacteria themselves and in the aquatic ecosystem is not well understood. The aim of this study is to evaluate if microcystins might be used as a communication tool for interspecies cyanobacterial communication via the promotion of oxidative stress. Reactive oxygen species (ROS) are known to be used as plant signals. The main questions relate to the promotion of oxidative stress in Synechocystis sp. via exposure to the cyanobacterial toxins and the physiological effects. This study shows a few markers for oxidative stress, such as the antioxidative enzymes superoxide dismutase, peroxidases and catalases, and cell damage due to extensive generation of ROS leading to lipid peroxidation. End products of lipid peroxidation (malonaldehyde and 4-hydroxynonenal) are conjugated by glutathione S-transferase.     

Distribution and toxicity of a new colonial Microcystis aeruginosa bloom in the San Francisco Bay Estuary,California

The first distribution, biomass and toxicity study of a newly established bloom of the colonial cyanobacteria Microcystis aeruginosa was conducted on October 15, 2003 in the upper San Francisco Bay Estuary. Microcystis aeruginosa was widely distributed throughout 180 km of waterways in the upper San Francisco Bay Estuary from freshwater to brackish water environments and contained hepatotoxic microcystins at all stations. Other cyanobacteria toxins were absent or only present in trace amounts. The composition of the microcystins among stations was similar and dominated by demethyl microcystin-LR followed by microcystin-LR. In situ toxicity computed for the >75 m cell diameter size fraction was well below the 1 g l^–1 advisory level set by the World Health Organization for water quality, but the toxicity of the full population is unknown. The toxicity may have been greater earlier in the year when biomass was visibly higher. Toxicity was highest at low water temperature, water transparency and salinity. Microcystins from the bloom entered the food web and were present in both total zooplankton and clam tissue. Initial laboratory feeding tests suggested the cyanobacteria was not consumed by the adult copepod Eurytemora affinis, an important fishery food source in the estuary.     

Blue green algal (cyanobacterial) toxins, surface drinking water, and liver cancer in Florida

The blue green algae or cyanobacteria represent a diverse group of organisms that produce potent natural toxins. There have been case reports of severe morbidity and mortality in domestic animals through drinking water contaminated by these toxins. Microcystins, in particular, have been associated with acute liver damage and possibly liver cancer in laboratory animals. Although, there has been little epidemiologic research on toxin effects in humans, a study by Yu (1995) found an association between primary liver cancer and surface water. Surface water drinking supplies are particularly vulnerable to the growth of these organisms; current US drinking water treatment practices do not monitor or actively treat for blue green algal toxins including the microcystins.After a monitoring survey in Florida found organisms and microcystins (among other cyanobacterial toxins) in surface water drinking sources, a pilot ecological study was performed using a Geographic Information System (GIS) to evaluate the risk of primary hepatocellular carcinoma (HCC) and proximity to a surface water treatment plant at cancer diagnosis. The study linked all HCC cancers diagnosed in Florida from 1981 to 1998 with environmental databases.A significantly increased risk for HCC with residence within the service area of a surface water treatment plant was found compared to persons living in areas contiguous to the surface water treatment plants. However, this increased risk was not seen in comparison to persons living in randomly selected ground water treatment service areas or compared to the Florida cumulative incidence rate for the study period, using various comparison and GIS methodologies. Furthermore, these findings must be interpreted in light of significant issues of latency, high population mobility, and the lack of individual exposure information. Nevertheless, the issue of acute and chronic human health effects associated with the consumption of surface waters possibly contaminated by blue green algal toxins merits further investigation.     

Bioaccumulation of Microcystins in Lettuce

The contamination of lettuce (Lactuca sativa L.) by water‐borne crude extracts of the cyanobacterium microcystin‐producing Microcystis aeruginosa (Kützing) Kützing was investigated. The aim of the study was to determine whether bioaccumulation of microcystins occurs in lettuce foliar tissue when sprayed with solutions containing microcystins at concentrations observed in aquatic systems (0.62 to 12.5 μg · L^?1). Microcystins were found in lettuce foliar tissues (8.31 to 177.8 μg per Kg of fresh weight) at all concentrations of crude extracts. Spraying with water containing microcystins and cyanobacteria may contaminate lettuce at levels higher than the daily intake of microcystins recommended by the World Health Organization (WHO), underscoring the need to monitor such food exposure pathways by public authorities.     

Can ingested cyanobacteria be harmful to the signal crayfish (Pacifastacus leniusculus)?

1. A sample of adult signal crayfish were taken from a pond with a hepatotoxic bloom of the benthic cyanobacterium Oscillatoria sancta . Cyanobacteria were found in the stomachs of thirty‐one out of thirty‐two crayfish examined.
2. To examine the effect of hepatotoxic cyanobacteria on crayfish a 14‐day feeding trial was carried out with thirty‐six animals. There were three treatments: (i) hepatotoxic and (ii) non‐toxic Planktothrix agardhii; and (iii) crayfish pellets as a control.
3. High‐performance liquid chromatography (HPLC) showed that microcystins (the toxins of P. agardhii ) had accumulated in the hepatopancreas of 50% of the animals in the toxic treatment.
4. The cyanobacteria did not appear to have any negative impact on the crayfish. All crayfish survived, remained motile and ate throughout the experiment.
5. During the experiment blood samples were taken and the total number of haemocytes counted. At the end of the experiment glucose concentration and relative wet weight of the hepatopancreas were measured. No differences between crayfish fed on toxic and non‐toxic P. agardhii and the controls were found.
6. The fact that microcystin accumulates in the crayfish hepatopancreas indicates that the toxin may be transferred further up the food chain.     

Genotoxicity of microcystic cyanobacteria extract of a water source in China.

The water pollution of toxic cyanobacteria (blue-green algae) is a worldwide problem and worsens with industrialization. Microcystins are potent cyclic heptapeptidic hepatotoxins produced mainly by Microcystis aeruginosa, and their hepatotoxicity has been well-documented. In contrast, information on the genotoxic effects of microcystins is relatively scarce. In our present study, the genotoxicity of microcystic cyanobacteria extract (MCE) of a water source in China was studied using Salmonella typhimurium assay (Ames test), comet assay (Single cell gel electrophoresis) and mouse micronucleus test. Results from Ames test indicated that MCE had strong mutagenicity regardless of the presence of S9. Moreover, MCE was able to induce DNA damage in primary cultured rat hepatocytes examined by comet assay. In addition, MCE also enhanced bone marrow micronucleated polychromatic erythrocytes in mice. The analysis of HPLC showed that the main component of MCE was microcystin-LR. The understanding of the potent genotoxicity of MCE will help to establish the possible link between water cyanobacteria contamination and high risk of primary liver cancer found in some endemic areas.     

Determination of microcystins in reservoirs of different basins in a semiarid area

Intracellular benthic microcystins and microcystins dissolved in water were detected in seven reservoirs in the Segura and Júcar basins located in a semiarid area in the province of Alicante (SE, Spain). Three varieties of intracellular benthic microcystins were identified: Mc-LR, Mc-RR and Mc-YR. Generally, all the microcystin varieties were present in the same reservoir and Mc-LR, considered the most powerful carcinogen known, predominated in most of them. Total microcystins absolute values per dry weight varied from 0.055 μg g^?1 in the Elx reservoir in summer to 1.032 μg g^?1 in the Crevillent reservoir in spring. Microcystins dissolved in water were not frequent in the reservoirs and were detected in Guadalest throughout the year, with values of between 0.041 and 0.069 ppb. In the Crevillent reservoir, dissolved microcystins were recorded only in summer with 0.163 ppb and were not recorded in the other reservoirs in any season. The cyanobacterial presence varied throughout the year, but no significant difference was seen for interseasonal microcystins production (ANOVA, p?>?0.1). The principal component analysis (PCA) and Pearson's correlation coefficient results showed that physico-chemical parameters did not significantly account for both intracellular and dissolved microcystins occurrence. Moreover, the PCA indicated that the evolution of the concentrations of dissolved and intracellular microcystins followed different patterns. The fact that benthic organisms could release intracellular toxins into water may be due to stress caused by changes in water levels and by colonies’ senescence. However, they may also perform antigrazing and/or allelopatic functions. A significant difference was found for conductivity, phosphates and the presence of microcystins Mc-LR and Mc-Tot in the oligo- and eutrophic reservoirs (t test, p?<?0.1 for all tests). In the reservoirs under study, microcystins production did not correlate with the high level of nutrients (t test, p?>?0.1). In fact, microcystins were never detected in the more eutrophic reservoirs, e.g. Tibi and Beniarrés, both of which correspond to the River Júcar basin. This fact indicates a significant difference for conductivity and the presence of microcystins Mc-RR (t test, p?<?0.1) between the River Segura basin and that of the River Júcar. The identification of microcystins in benthic cyanobacteria from the less eutrophic semiarid studied reservoirs showed that hepatotoxin production was restricted to neither planktonic species nor the physico-chemical parameters considered typical for the occurrence of cyanobacterial toxins.     

Occurrence of toxigenic cyanobacterial blooms in freshwaters of Sri Lanka

A previous pioneering study of freshwater bodies in Sri Lanka revealed the presence of toxic cyanobacteria in three out of four water bodies tested. It was therefore important to perform a more detailed investigation into the presence of cyanobacteria and their toxins throughout Sri Lanka. The country has a long history of well-planned water management with the agricultural economy and drinking water supply still dependent on thousands of man-made tanks. Seventeen reservoirs from different user categories and different climatic zones were selected to study variations in phytoplankton communities with relation to major nutrients, with particular emphasis on cyanobacteria. The study was carried out during a two-year period and heavy growths or blooms of cyanobacteria observed during the study period were tested for microcystins. The results clearly categorised the 17 reservoirs into four groups parallel to the classification based on the user categories of water bodies. Biomass of total phytoplankton, the abundance of cyanobacteria, the dominance of Microcystis spp. and concentration of nitrate (N) and total phosphorous (P) were the lowest in drinking water bodies and the highest in aesthetic water bodies. Irrigation water bodies showed the second lowest values for phytoplankton biomass, and concentration of N and P, while hydropower reservoirs showed the second highest values for the same parameters. The fraction of cyanobacteria in irrigation waters was higher than that in hydropower reservoirs, but surprisingly the dominance of Microcystis spp. was reversed. Possible reasons for these variations are discussed. More than half of the bloom material tested contained microcystins up to 81microgl(-1). Our findings indicate the potential for high-risk situations due to toxigenic cyanobacterial blooms in susceptible freshwaters of Sri Lanka.     

微囊藻毒素对陆生植物的污染途径及累积研究进展

微囊藻毒素(Microcystins,MCs)是全世界范围内普遍存在、且随着水体污染的加剧而在自然环境中大量积聚的蓝藻毒素之一,对多种生物有着严重的毒性作用.MCs在生物体内富集并通过食物链传递,对人类健康造成威胁.近些年,MCs对陆生植物的毒害作用及累积研究尤为引人关注,取得了一批重要的研究成果.MC-LR(L为亮氨酸)和MC-RR(R为精氨酸)是淡水水体中普遍存在且危害较大的两种MCs异构体.针对这两种毒素,重点介绍其对陆生植物的污染途径、毒性作用及其在作物体内的累积量,对今后的研究进行了展望.     

Extraction and analysis of microcystins RR and LR in cyanobacteria using a cyano cartridge

A new method for the extraction of microcystins RR and LR in cyanobacteria was developed using a cyano cartridge. Lyophilized cells (100 mg) were extracted with 5% (v/v) acetic acid. The extract was centrifuged and then the supernatant was applied to a CN cartridge. The cartridge that contained microcystins was rinsed with 5 ml of water and 5 ml of 0.5 M acetic acid, followed by 5 ml of 5% acetonitrile in water. Microcystins were finally eluted from the CN cartridge with 70% acetonitrile in water and were determined by HPLC. Better recoveries and chromatograms were observed than with ODS cartridges.     

Investigation of the effect of exposure to non cytotoxic amounts of microcystins

This paper describes a metabolomic approach for investigation of the potential effect of exposure of humans to low amounts of microcystins using HepG2 cell line. Microcystins are hepatotoxins produced by cyanobacteria (blue-green algae) which occur in water bodies with high eutrophication especially those with a slow flow rate or those that are stagnant in warm climates. Mammalian exposure to these compounds has been associated with deleterious effects and in high dosage cases, deaths of animals has been reported. The metabolic profile of HepG2 cells is closely related to that of hepatocytes and therefore serves as a good model due to their human origin. Proton nuclear magnetic resonance spectroscopy (¹H NMR) and direct injection mass spectrometry (DIMS) were used to analyse media extracts from the cells and data obtained was reduced by chemometric methods. The use of principal component analysis (PCA) enabled achievement of a visual distinction between the metabolic profiles of samples exposed to microcystins, control samples (unexposed), and those which were exposed to acetaminophen (positive control). A profile of media components showed that some components in the samples exposed to microcystins increased compared to those in control samples. They included amino acids, organic acids, lipids, some purines and pyrimidines. In general exposure to low concentration of microcystin was found to interfere with amino acid metabolism, carbohydrate metabolism, lipid metabolism and nucleic acids metabolism. Furthermore, low concentration of microcystins did not result in significant cell death; rather the cells continued to proliferate.     

Early biochemical effects of Microcystis aeruginosa extracts on juvenile rainbow trout (Oncorhynchus mykiss)

Microcystins (MC) are usually the predominant cyanotoxins associated with cyanobacterial blooms in natural surface waters. These toxins are well-known hepatotoxic agents that proceed by inhibiting protein phosphatase in aquatic biota; recent studies have also reported oxidative stress and disruption of ion regulation in aquatic organisms. In the present study, young trout (Oncorhynchus mykiss) were exposed to crude extracts of Microsystis aeruginosa for four days at 15 °C. The level of microcystins was calculated to confirm the presence of toxins in these crude extracts: 0, 0.75, 1.8 and 5 μg/L. Protein phosphatase measured in the liver increased by at least 3-fold and is significantly as a result of exposure to these sublethal concentrations of crude extract, his indicates an early defense response against protein phosphatase inhibition from cyanotoxins. This was corroborated by the decreased phosphate content in proteins found in the liver and brain. No increase in glutathione-S transferase (GST) activity was observed and lipid peroxidation was unaffected in both liver and brain tissue exposed to the cyanobacterial extracts. The data revealed that the proportion of the reduced (metal-binding) form of metallothionein (MT) decreased by two-fold relative to the control group (with a concomitant increase in the proportion of the oxidized form). The level of phosphate associated with MT increased by 1.5-fold at the highest concentration of crude extract. Acetylcholinesterase (AChE) activity in brain tissue was decreased after exposure to the highest concentration of crude extract, suggesting a slowdown in neural activity. However, no biotransformation processes or detoxification of GST was triggered. Our findings show early sign of biochemical effects of MC-LR in young trout.     

含微囊藻毒素的蓝藻水华提取物对小鼠抗氧化酶的毒性作用

在世界范围内发生的富含营养的废水中生长蓝藻水华已经造成了严重的水污染。已有报道显示水华中的有毒成分是一类单环七肽微囊藻毒素,它们对人和家畜有严重的毒害作用,本文所使用的蓝藻水华采集于我国的太湖地区。我们采用三个亚致死计量(16、32、64 mg冻干藻细胞/kg体重,相当于4.97、9.94、19.88μg藻毒素/kg体重)的蓝藻水华提取物(CBE),分析它们对小鼠肝脏抗氧化酶的影响。经过连续14d每天腹腔注射CBE,小鼠分别在第3、5、7、9、11、13、15天处死并检测三种主要的抗氧化酶:超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽巯基转移酶(GST)的活性。SOD和CAT的活性均呈现了抑制效应,GST的活性也发生了明显的变化。为了进一步确定过氧化的程度,我们又通过检测丙二醛的含量分析了脂质过氧化的程度。经过14d CBE处理后脂质过氧化表现出了明显的浓度依赖性的上升趋势。我们的结果表明,CBE对小鼠的抗氧化酶有严重毒害作用,并且能够引起脂质的过氧化。     

Effects of microcystins over short- and long-term memory and oxidative stress generation in hippocampus of rats

Microcystins produced by cyanobacteria are potent inhibitors of some protein phosphatases, but recent evidence also indicates its potential to generate oxidative stress. In the present study, the effects of microcystin raw extracts (Mic; 0.01 and 20microg/L) and purified okadaic acid (OA; 0.01 and 10microg/L) on short- and long-term memory alteration and antioxidant and oxidative damage were investigated in hippocampus of rats. The results showed an amnesic effect with 0.01 and 20microg/L Mic on retrieval and only with 0.01microg/L Mic on spatial learning. Parallel to these effects oxidative damage was observed as evidenced by augmented levels of lipid peroxides and DNA damage and the absence of antioxidant responses in terms of total oxyradical scavenging capacity. Phase II reactions catalyzed by glutathione-S-transferase were not modified after microcystins exposure. Overall this study showed physiological events (retrieval and spatial learning) that can be related to the classical toxic effects of microcystins (i.e., phosphatase inhibition). In addition, evidence of alternative toxicity mechanisms via oxidative stress generation was also obtained. The fact that organic anion transporter polypeptides (OATP) involved in microcystins uptake are expressed not only in liver but also in brain points to the environmental relevance of the observed effects.     

Release of heptapeptide toxin (microcystin) during the decomposition process of Microcystis aeruginosa.

The decomposition process of toxic blue-green alga (cyanobacteria), Microcystis aeruginosa, under dark and aerobic condition was investigated in relation to the change of the amounts of heptapeptide toxins (microcystins YR and LR) by two experiments: one with Microcystis cells and the other with two purified microcystins. In the experiment with Microcystis cells, an increase of heterotrophic bacteria observed from the beginning of the experiment, was followed by decomposition of the algal cells and the subsequent release of microcystins into the filtrate fraction. The amounts of the toxins initially present in the cells were quantitatively detected in the filtrate fraction on the 35th day. The decomposition of microcystin YR began on the 42nd day. The decomposition rate of the two toxins was different. The decomposition rate of purified microcystins YR and LR, compared in distilled water and culture medium, respectively, indicated clearly that microcystin YR was more labile to decomposition than microcystin LR in the culture medium. At the end of the experiment (45th day) microcystin YR decreased to 58.6%, while 86.2% of microcystin LR remained.     

Response of Daphnia's Antioxidant System to Spatial Heterogeneity in Cyanobacteria Concentrations in a Lowland Reservoir

Many species and clones of Daphnia inhabit ecosystems with permanent algal blooms, and they can develop tolerance to cyanobacterial toxins. In the current study, we examined the spatial differences in the response of Daphnia longispina to the toxic Microcystis aeruginosa in a lowland eutrophic dam reservoir between June (before blooms) and September (during blooms). The reservoir showed a distinct spatial pattern in cyanobacteria abundance resulting from the wind direction: the station closest to the dam was characterised by persistently high Microcystis biomass, whereas the upstream stations had a significantly lower biomass of Microcystis. Microcystin concentrations were closely correlated with the cyanobacteria abundance (r = 0.93). The density of daphniids did not differ among the stations. The main objective of this study was to investigate how the distribution of toxic Microcystis blooms affects the antioxidant system of Daphnia. We examined catalase (CAT) activity, the level of the low molecular weight antioxidant glutathione (GSH), glutathione S-transferase (GST) activity and oxidative stress parameters, such as lipid peroxidation (LPO). We found that the higher the abundance (and toxicity) of the cyanobacteria, the lower the values of the antioxidant parameters. The CAT activity and LPO level were always significantly lower at the station with the highest M. aeruginosa biomass, which indicated the low oxidative stress of D. longispina at the site with the potentially high toxic thread. However, the low concentration of GSH and the highest activity of GST indicated the occurrence of detoxification processes at this site. These results demonstrate that daphniids that have coexisted with a high biomass of toxic cyanobacteria have effective mechanisms that protect them against the toxic effects of microcystins. We also conclude that Daphnia''s resistance capacity to Microcystis toxins may differ within an ecosystem, depending on the bloom''s spatial distribution.