微囊藻毒素免疫检测技术研究进展

微囊藻毒素主要是由蓝绿藻属产生的的一类毒素,其毒性主要表现在特异性抑制细胞内的蛋白磷酸酶(Ppl和PP2A)。目前有关微囊藻毒素分析和检测的研究开展得十分广泛,免疫检测技术由于其众多的优点更显示出其良好的应用前景。本文详细地综述了目前国内外在微囊藻毒素免疫检测方面的研究方法和成果;并在总结这些研究的基础上,展望了微囊藻毒素免疫检测技术的发展方向。     

Inhibition of embryonic development by microcystin-LR in zebrafish, Danio rerio.

Microcystin-LR (MC-LR), a cyanobacterial toxin, is a potent inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A). PP1 and PP2A are critical regulators in embryonic development. However, the effects of MC-LR in embryonic development have been controversial. MC-LR has been demonstrated to be highly toxic in medaka, but not in zebrafish or rabbit embryos. The causes of difference may be due to membrane impermeability that impaired the delivery of MC-LR into cytoplasm of zebrafish and rabbit embryos. Therefore, we microinjected MC-LR directly into developing zebrafish embryos and investigated the effects of MC-LR on embryonic development. We demonstrated that MC-LR induced the lethality of zebrafish embryos in a dose- and time-dependent manner. MC-LR also induced the loss of blastomere coherence via the interference of beta-catenin and cadherins distributions. Furthermore, the MC-LR treated fry revealed various developmental defects. These results suggested that MC-LR might affect the phosphorylation equilibrium of signaling molecules, including beta-catenin and cadherins, required early in zebrafish embryonic development.     

Identification and Determination of Microcystins in Source Water an Waterbloom Sample From Meiliang Bay, Taihu Lake, China

OBJECTIVE: To identify and determine the congener and level of microcystins in the source water of Taihu Lake. METHODS: Improved method of SPE combined with HPLC was employed to detect the concentration and varieties of microcystins in source water and bloom samples collected from Meiliang Bay, Taihu Lake. RESULTS: The contents of two predominant microcystin components, MC-RR, and MC-LR, were relatively high in samples during warm months and correlated with the phase of algae growth. The maximum concentrations of MC-RR and MC-LR in water sample reached 3.09 +/- 0.53 microg/L and 2.39 +/- 0.41 microg/L during the period of water bloom in September 2004, respectively. Even without waterbloom, the concentration of MC-LR in source water sample was still higher than the guideline value. CONCLUSION: The status of microcystin pollution in this region is serious and measures to monitor and control the growth of cyanobacteria are urgently needed.     

Detection of microcystin synthetase genes in health food supplements containing the freshwater cyanobacterium Aphanizomenon flos-aquae.

In this study we investigated the presence of toxin-producing cyanobacterial contaminants in food supplements manufactured from blooms of the non-toxic freshwater cyanobacterium Aphanizomenon flos-aquae. Previous reports investigating the contamination of health food supplements with toxin-producing cyanobacteria have used chemical and or biochemical methods such as HPLC, ELISA and protein phosphatase assays. Whilst these studies have drawn attention to the presence of hepatotoxic microcystins in some commercially available food supplements, the methods used do not provide any information on the source of the contaminant. Such information would be useful for the quality control of food supplements produced for human consumption. In this study we applied a molecular technique, involving the amplification of the 16s rRNA gene, the phycocyanin operon, and two genes of the microcystin synthetase gene cluster to show that all 12 food supplement samples, sourced from various internet distributors and containing non-toxic A. flos-aquae, also contained toxigenic cyanobacteria. Sequencing of the microcystin synthetase genes detected in all of the food supplements showed that M. aeruginosa was the organism responsible for the production of microcystins in the samples. The presence of microcystins in the food supplements was confirmed by ELISA, with concentrations within the range of 0.1--4.72 microgg(-1) (microcystin-LR equivalents). Given that the molecular methods applied here are highly sensitive, and show good agreement with the results obtained from ELISA, we believe that they could potentially be used as a quality control technique for food products that contain cyanobacteria.     

Occurrence and elimination of cyanobacterial toxins in two Australian drinking water treatment plants.

In Australian freshwaters, Anabaena circinalis, Microcystis spp. and Cylindrospermopsis raciborskii are the dominant toxic cyanobacteria. Many of these surface waters are used as drinking water resources. Therefore, the National Health and Medical Research Council of Australia set a guideline for MC-LR toxicity equivalents of 1.3 microg/l drinking water. However, due to lack of adequate data, no guideline values for paralytic shellfish poisons (PSPs) (e.g. saxitoxins) or cylindrospermopsin (CYN) have been set. In this spot check, the concentration of microcystins (MCs), PSPs and CYN were determined by ADDA-ELISA, cPPA, HPLC-DAD and/or HPLC-MS/MS, respectively, in two water treatment plants in Queensland/Australia and compared to phytoplankton data collected by Queensland Health, Brisbane. Depending on the predominant cyanobacterial species in a bloom, concentrations of up to 8.0, 17.0 and 1.3 microg/l were found for MCs, PSPs and CYN, respectively. However, only traces (<1.0 microg/l) of these toxins were detected in final water (final product of the drinking water treatment plant) and tap water (household sample). Despite the low concentrations of toxins detected in drinking water, a further reduction of cyanobacterial toxins is recommended to guarantee public safety.     

Bacterial degradation of microcystin toxins in drinking water eliminates their toxicity.

Microcystin-LR and -LA were readily biodegraded by a bacterium, Sphingpoyxis sp. LH21, in a treated reservoir water. Detection of the microcystins was conducted using high-performance liquid chromatography (HPLC), protein phosphatase 2A (PP2A) inhibition assay and a cell-based cytotoxicity assay. The HPLC results correlated well with the two assays. The decrease in cytotoxicity, coupled with the associated decrease in microcystin concentrations, indicated that no cytotoxic by-products were being generated, highlighting the applicability of biodegradation as a feasible treatment option for effective microcystin removal.     

Detection of microcystin–metal complexes by using cryospray ionization-Fourier transform ion cyclotron resonance mass spectrometry

Complexes of microcystins (MCs) and metal ions were detected with mass spectrometry. To observe the MCs-metals complexes, CryoSpray ionization ion source was developed and it was equipped on a commercial Fourier transform ion resonance mass spectrometer. Mixtures of two MCs and five metals were applied to Fourier transform ion cyclotron resonance mass spectrometry coupled with CryoSpray. The analyses showed complexes of MC-LR-Fe(II), -Zn, -Cu, -Mg, MC-RR-Fe(II), -Zn, -Cu, and -Mg but MC-LR-Fe(III) and MC-RR-Fe(III) were not observed. Present study suggested that MCs may play roles in metal ion uptake and/or accumulation in the algal cells.     

太湖水域及鱼类体内微囊藻毒素的调查

目的 了解太湖水中微囊藻毒素(MC)的污染情况.方法 于2001年7、11月采集太湖水样12件,每件50ml,装入灭菌的塑料瓶中;捕捉鲤形目和鲶形目的主要鱼种7属,共计28尾.用ELISA法对太湖源水的水样和鱼样进行MC含量的检测.结果 太湖水样均检出MC.7月水样MC含量均值为9874.8pg/ml,高于11月(142.8pg/ml),差异有统计学意义(t=3.305,P=0.003).主要鱼类体内均检出MC.其中,肝脏中MC的含量均值为27.84ng/g,高于肌肉中MC的含量均值(3.97ng/g),差异有统计学意义(t=5.933,P=0.001).结论 太湖水域中的自然水体和鱼体内已不同程度地含有MC.     

Genetic contributions to the risk assessment of microcystin in the environment

Of the known toxins produced by cyanobacteria, microcystins and nodularins are the most significant threat to human and animal health. Knock-out studies have confirmed that microcystins are produced nonribosomally by a multienzyme complex consisting of peptide synthetases, polyketide synthases, and tailoring enzymes. Gene clusters for microcystin biosynthesis have been identified and sequenced in the distantly related cyanobacterial genera Microcystis, Planktothrix, and Anabaena. Homologous genes have been detected in a nodularin-producing Nodularia strain. Subsequently, microcystin biosynthesis (mcy) genes have been used to establish molecular techniques for the detection of toxigenic cyanobacteria in laboratory and field studies. mcy genes of unknown origin can be assigned to the producing species. Techniques are currently being developed for the quantification of mcy genes in field populations. These initial genetic investigations pave the way for a molecular monitoring of microcystin- and nodularin-producing cyanobacteria and for studying the dynamics of toxic cyanobacteria in lakes. Furthermore, microcystin-deficient mutants have significantly increased our knowledge about the impact of the toxins on Microcystis-Daphnia interactions. The experience gained on microcystin biosynthesis genes will be valuable for a risk assessment of microcystin in the environment and for future water management and lake-restoration strategies.     

绿茶茶多酚对微囊藻毒素诱导生长调控原癌基因表达的抑制

[目的]通过观察微囊藻毒素对生长调控原癌基因（growth-regulate oncogene，GRO）的调控以及茶多酚对其拮抗作用，以探讨微囊藻毒素致癌机制。[方法]用Northem印记法及酶免疫法测定生长调控原癌基因的表达及蛋白浓度。[结果]实验发现NIH-313细胞在正常情况下并无生长调控原癌基因表达。微囊藻毒素能够强烈诱导GRO的表达和分泌，并且这种诱导呈现浓度和时间依赖的方式。不同浓度的茶多酚能够对微囊藻毒素诱导的GRO的表达和分泌进行有效抑制，并且这种抑制随时间而加强。[结论]茶多酚抑制微囊藻毒素对GRO的表达和分泌的诱导。本文的发现为进一步了解微囊藻毒素的致癌性，以及茶多酚的抗癌机制和应用提供了新的方向。