Tissue distribution of microcystin (MC)-LR-GSH, MC-LR-Cys and MC-LR of omnivorous fish in Lake Taihu was investigated. MC-LR and MC-LR-Cys were detected in liver, kidney and muscle. The concentration of MC-LR in liver and kidney was 0.052 ug g-1 DW and 0.067 ug g-1 DW, respectively. MC-LR-Cys appeared to be an important metabolite with average contents of 1.104 ug g-1 DW and 0.724 ug g-1 DW in liver and kidney, and the MC-LR-Cys/MC-LR ratio in liver and kidney reaching as high as 21.4 and 10.8. High MC-LR-Cys/MC-LR ratio and a significant correlation between MC-LR-Cys and MC-LR concentration in liver, suggest that liver is more active in detoxification of MC-LR by formation of MC-LR-Cys for omnivorous fish. Furthermore, there might be a balance between the accumulation and depuration/metabolism of MC-LR-Cys in kidney. The MC-LR-Cys can be formed in kidney directly, or transported from liver or other tissues, while the MC-LR-Cys in kidney might be dissociated to MC-LR or excreted. Although MC-LR and its metabolites were scarcely detected in muscle, it is necessary to investigate the distribution of toxic metabolites in edible muscle.
The liver plays a large role in the detoxification of microcystins. Detoxification products were seen in urine, feces, and liver cytosolic fractions, but these products have not been structurally identified. The detoxification products of microcystin-LR are more water soluble than the parent toxin.
Glutathione (GSH) plays crucial roles in antioxidant defense and detoxification metabolism of microcystin-LR (MC-LR). However, the detoxification process of MC-LR in mammals remains largely unknown. This paper, for the first time, quantitatively analyzes MC-LR and its GSH pathway metabolites (MC-LR-GSH and MC-LR-Cys) in the liver of Sprague-Dawley (SD) rat after MC-LR exposure. Rats received intraperitoneal (i.p.) injection of 0.25 and 0.5 lethal dose 50 (LD50) of MC-LR with or without pretreatment of buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH synthesis. The contents of MC-LR-GSH were relatively low during the experiment; however, the ratio of MC-LR-Cys to MC-LR reached as high as 6.65 in 0.5 LD50 group. These results demonstrated that MC-LR-GSH could be converted to MC-LR-Cys efficiently, and this metabolic rule was in agreement with the data of aquatic animals previously reported. MC-LR contents were much higher in BSO+MC-LR-treated groups than in the single MC-LR-treated groups. Moreover, the ratio of MC-LR-Cys to MC-LR decreased significantly after BSO pretreatment, suggesting that the depletion of GSH induced by BSO reduced the detoxification of MCs. Moreover, MC-LR remarkably induced liver damage, and the effects were more pronounced in BSO pretreatment groups. In conclusion, this study verifies the role of GSH in the detoxification of MC-LR and furthers our understanding of the biochemical mechanism for SD rats to counteract toxic cyanobacteria.
来源:Hazardous Substances Data Bank (HSDB)
代谢
在当前工作中，已经扩展了关于人类MC代谢的现有信息，并在人类肝脏细胞溶质中确认了人类GST与MC-LR结合的能力。在生理GSH含量下，自发性反应占主导地位，而在GSH耗尽后，酶促反应占主导地位；这种转变在较高的GSH水平下是可以检测到的，MC浓度越低，这种转变越明显。然而，在低MC-LR浓度（=10 uM）下，代表反复口服暴露的情况下，酶促反应的重要性在GSH浓度在1到2 mM之间时变得占主导地位。MC-LR结合物在=0.5 mM GSH时可以被检测到，而对于10 uM MC-RR，在0.05 mM GSH时就可以观察到可检测水平的结合物，这个浓度比MC-LR低10倍。总的来说，我们的数据表明，MC-RR比MC-LR更有效地结合，尤其是在低浓度下。使用大鼠和小鼠的细胞溶质样本对MC-LR和MC-RR的GSH结合进行了表征，并检查了可能的物种差异。在生理GSH含量下，两种啮齿动物物种的酶促反应占总结合物形成的一半，与人类相比，减少了自发性反应的影响。大鼠和小鼠的GST显示出与人类的MC-LR和-RR GSH结合相似，但催化效率比人类样本高两倍。这主要是由于对底物的亲和力更高，动物模型中的Kmapp值比人类肝脏细胞溶质低一个数量级。与人类相比，两种变异体在啮齿动物中的代谢差异更为显著。
The accepted pathway for MC biotransformation is GSH conjugation, occurring either spontaneously or catalyzed by GST. In the present work, the already available information on human MC metabolism have been expanded and the capacity of human GST to conjugate MC-LR has been confirmed in human liver cytosol. At physiological GSH content the spontaneous reaction predominated on the enzymatic one; the prevalence of the enzymatic reaction occurred following GSH depletion, and the shift was detectable at higher GSH levels, the lower was MC concentration. However, at low MC-LR concentrations (=10 uM), representative of repeated oral exposure, the relevance of the enzymatic reaction became predominant at GSH concentration between 1 and 2 mM. MC-LR conjugate was detectable at =0.5 mM GSH, whereas, with 10 uM MC-RR detectable levels of conjugate were observed at 0.05mM GSH, a 10-fold lower concentration. Overall, our data indicate that MC-RR is more efficiently conjugated than MC-LR, especially at low concentrations. Cytosol samples from rat and mouse were used to characterize GSH conjugation of MC-LR and MC-RR, and to check for possible species differences. At physiological GSH content, in both rodent species the enzymatic reaction accounted for half of the total conjugate formation, reducing the impact of spontaneous reaction with respect to human. Rat and mouse GST showed similar MC-LR and-RR GSH conjugation, but a two-fold higher catalytic efficiency than human sample. This is mainly due to higher affinity for the substrate, with Kmapp values being an order of magnitude lower in the animal models than in human liver cytosol. More pronounced differences in the metabolism of the two variants were evidenced in rodents than in humans.
Microcystins are extremely stable and resist common chemical breakdown such as hydrolysis or oxidation under conditions found in most natural water bodies. These toxins can break down slowly at high temperature (40 C or 104 F ) at either very low (<1) or high (>9) pH. The half-life, the time it takes for one-half of the toxin to degrade, at pH 1 and 40 oC is 3 weeks; at typical ambient conditions half-life is 10 weeks.
IDENTIFICATION AND USE: Microcystin-LR (MCLR) is the most investigated cyanobacterial peptide toxin because it is frequently present in cyanobacterial blooms in rivers and lakes. Cyanobacterial toxins are classified by how they affect the human body: Hepatotoxins affect the liver and are produced by some strains of Microcystis, Anabaena, and Oscillatoria. They consist of cyclic hetapepides, termed microcystins and nodularins, which are cyclic pentapeptides. HUMAN STUDIES: Through the recreational use of contaminated water, cyanobacterial blooms of Microcystis, Anabaena, and others have been linked to incidence of human illness in many countries. In Canada, human illnesses have been reported in Saskatchewan, with symptoms including stomach cramps, vomiting, diarrhea, fever, headache, pains in muscles and joints, and weakness. Similar symptoms as well as skin, eye, and throat irritation and allergic responses to cyanobacterial toxins in water have also been reported in other countries. An outbreak of acute liver failure occurred at a dialysis center in Caruaru, Brazil. At the clinic, 116 (89%) of 131 patients experienced visual disturbances, nausea, and vomiting after routine hemodialysis treatment on 13-20 February 1996. Subsequently, 100 patients developed acute liver failure, and many of them died. From liver concentrations and exposure volumes, it was estimated that 19.5 ug/L microcystin was in the water used for dialysis treatments. MCLR had a clastogenic effect in human lymphoblastoid cell line. The analysis of the micronucleus content in HepG2 cells suggested that MCLR induces both chromosome breaks and loss. MCLR induced cell death in primary human hepatocytes in vitro. ANIMAL STUDIES: The microcystins are primarily hepatotoxins. After acute exposure by iv or ip injection of microcystin, severe liver damage is characterized by a disruption of liver cell structure (due to damage to the cytoskeleton), a loss of sinusoidal structure, increases in liver weight due to intrahepatic hemorrhage, hemodynamic shock, heart failure and death. Other organs affected are the kidneys and lungs and the intestines. MCLR-induced hepatotoxicity occurs through specific inhibition of serine/threonine protein phosphatases 1 and 2A, which leads to hyperphosphorylation of many cellular proteins. This eventually results in cytoskeletal damage, loss of cell morphology, and the consequent cell death. Inhibition of protein phosphatases is the main mechanism associated with the potential tumor-promoting activities of MCLR. It can induce excessive formation of reactive oxygen and nitrogen species, which results in DNA damage. Although MCLR is not a bacterial mutagen, in mammalian cells it can induce mutations, as predominantly large deletions, and it has clastogenic actions. MCLR also disrupts the mitotic spindle, and interferes with DNA damage repair processes, which contribute to genetic instability. Furthermore, MCLR increases expression of early response genes, including proto-oncogenes, and genes involved in responses to DNA damage and repair, cell-cycle arrest, and apoptosis. In rats oral exposure to MCLR had adverse affects on neurobehaviors, and induced inflammation in memory-related brain regions. MCLR induced developmental neurotoxicity in zebrafish. In mice, perinatal MCLR exposure interfered with the development of the prostate in the offspring, evidenced by prostatic necrosis, hyperplasia, inflammation, and fibrosis. Exposure to MCLR in zebrafish results in the disturbance of thyroid hormone homeostasis. MCLR significantly impaired the spermatogenesis in mice possibly through the effect on Hypothalamic-Pituitary-Gonadal Axis. MCLR crosses the blood-testis barrier and interferes with DNA damage repair pathway and also increases expression of the proto-oncogenes in testis. MCLR treated mice exhibit oxidative stress in testis through the alteration of antioxidant enzyme activity. ECOTOXICITY STUDIES: Cyanobacterial water blooms can significantly alter the normal development of amphibian embryos. Low concentrations of dissolved microcystin had no harmful effect on Daphnia. On the contrary, stimulatory effects were detected. With microcystin given at a higher dosage or for a longer exposure, the effect on Daphnia magna was fatal. Acute MCLR exposure has the potential to disturb the homeostasis of thyroid hormone metabolism, leading to a hypothyroidism state in the juvenile Chinese rare minnow. MCLR at environmentally relevant concentrations is able to induce impairments on fish gonadal development in zebrafish. In C. elegans, MCLR induced adverse effects on spermiogenesis. MCLR could affect terrestrial plants seedling germination and growth.
来源:Hazardous Substances Data Bank (HSDB)
毒理性
毒性总结
微囊毒素的作用部位是肝细胞，这是肝脏中最常见的细胞类型。它们通过破坏细胞骨架起作用，细胞骨架是一种适应性蛋白框架，它不断地塑造和重塑细胞，以响应环境。细胞死亡，这会破坏肝脏的细小血管，导致大量肝出血。
其分子靶标是一组称为蛋白磷酸酶的酶，它们在调节蛋白相互作用和活性方面发挥作用。非常明确的蛋白磷酸酶类型（1型和2A型）会被极低浓度的微囊毒素非常特异地抑制。这种酶从蛋白中去除磷酸，这是许多生化途径中的常见步骤。这种抑制，以及随后磷酸化蛋白的积累，被认为是微囊毒素破坏肝脏的机制之一。
微囊毒素还能激活磷酸化酶b，这在肝细胞的事务中起着非常重要的作用。这种抑制和激活的结合对细胞来说是迅速致命的。这些毒素中的一些特异性使它们成为有价值的 research 工具。
The site of action of microcystins is the hepatocyte, the commonest cell type in the liver. They act by disrupting the cytoskeleton, the adaptable protein framework that constantly shapes and reshapes the cell as it responds to the environment. The cells die and this destroys the finer blood vessels of the liver leading to massive hepatic bleeding.
The molecular target are a group of enzymes called protein phosphatases that play a role in regulating protein interactions and activities. Very well-defined types of protein phosphatase (type 1 and type 2A) are inhibited very specifically by very low concentrations of microcystins. This enzyme removes phosphate from a protein, a common step in many biochemical pathways. This inhibition, with
subsequent build up of phosphorylated proteins, is believed to be a mechanism by which microcystins destroy livers.
Microcystins also activate the enzyme phosphorylase b, which plays a very important role in the affairs of the hepatocyte. The combination of inhibition and activation is rapidly lethal to the cell. The specificity of some of these toxins makes them valuable research tools.
Evaluation: There is inadequate evidence in humans for the carcinogenicity of microcystin-LR ... There is inadequate evidence in experimental animals for the carcinogenicity of microcystin-LR. There is inadequate evidence in experimental animals for the carcinogenicity of Microcystis extracts ... Overall evaluation: Microcystin-LR is possibly carcinogenic to humans (Group 2B). Microcystis extracts are not classifiable as to their carcinogenicity to humans (Group 3) ...
来源:Hazardous Substances Data Bank (HSDB)
毒理性
致癌物分类
国际癌症研究机构致癌物：微囊藻毒素-LR
IARC Carcinogenic Agent:Microcystin-LR
来源:International Agency for Research on Cancer (IARC)
毒理性
致癌物分类
国际癌症研究机构（IARC）致癌物分类：2B组：可能对人类致癌
IARC Carcinogenic Classes:Group 2B: Possibly carcinogenic to humans
来源:International Agency for Research on Cancer (IARC)
The most likely route of exposure to cyanobacterial toxins is via oral ingestion. However, there have been no pharmacokinetic studies with orally administered microcystins. After intravenous or intraperitoneal injection of sublethal doses of variously radiolabelled toxins in mice and rats, microcystin appears to be transported by bile acids transporter in both the intestine and the liver. About 70% of the toxin is rapidly localized in the liver. The kidney and intestine also accumulate significant amounts of microcystin-LR. Microcystin-LR was excreted rapidly, with 75% of the total excretion occurring within 12 hours. The remaining 24% of the administered dose was excreted after 6 days, about 9% via the urinary route and 15% slowly (1% per day) via the fecal route.
Microcystin-LR does not readily cross cell membranes, and hence does not enter most tissues. After oral uptake it is transported across the ileum into the bloodstream through a bile acid type transporter (the multispecific organic ion transport system) present in hepatocytes and cells lining the small intestine and is concentrated in the liver as a result of active uptake by hepatocytes. It is covalently bound to a 40 kdalton protein (protein phosphatase 2A and possibly protein phosphatase 1) in the hepatocyte cytosol. Some other microcystin congeners are more hydrophobic than microcystin-LR and may cross cell membranes by other mechanisms, including diffusion.
... Serum samples /were analyzed/ using enzyme-linked immunosorbent assay (ELISA) methods to detect free microcystins, and gas chromatography/mass spectrometry (GC/MS) to detect 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB). MMPB is derived from both free and protein-bound microcystins by chemical oxidation, and it appears to represent total microcystins present in serum. ... Evidence of free microcystins /was found. in patient serum for more than 50 days after the last documented exposure. Serum concentrations of free microcystins were consistently lower than MMPB quantification of total microcystins: free microcystins as measured by ELISA were only 8-51% of total microcystin concentrations as detected by the GC/MS method. After intravenous exposure episodes, we found evidence of microcystins in human serum in free and protein-bound forms, though the nature of the protein-bound forms is uncertain. Free microcystins appear to be a small but variable subset of total microcystins present in human serum... /Microcystins/
The distribution, excretion and hepatic metabolism of (3H)microcystin-LR (sublethal iv) were measured in mice. Plasma elimination was biexponential with alpha- and beta-phase half-lives of 0.8 and 6.9 min, respectively. At 60 min, liver contained 67 +/- 4% of dose. Through the 6-day study the amount of hepatic radioactivity did not change whereas 23.7 +/- 1.7% of the dose was excreted; 9.2 +/- 1.0% in urine and 14.5 +/- 1.1% in feces. Approximately 60% of the urine and fecal radiolabel 6 and 12 hr postinjection was the parent toxin. Hepatic cytosol, which contained 70 +/- 2% of the hepatic radiolabel (1 hr through 6 days), was prepared for high-performance liquid chromatography analysis by heat denaturation, pronase digestion and C18 Sep Pak extraction. At 1 hr, 35 +/- 2% of the radiolabel was insoluble or C18 Sep Pak-bound; 43 +/- 3% was associated with a peak of retention time (rt) 6.6 min, and 16 +/- 3% with the parent toxin (rt 9.4 min). After 6 days, 8 +/- 1% was C18 Sep Pak-bound or insoluble; 5 +/- 0% occurred at rt 6.6 min, 17 +/- 1% with parent and 60 +/- 2% was associated with rt 8.1 min. Two other peaks, rt 4.9 and 5.6 min, appeared transiently. Analysis of hepatic cytosol by desalting chromatography under nondenaturing and denaturing conditions revealed that all of the radiolabel was associated with cytosolic components, and 83 +/- 5% was bound covalently through 1 day. By day 6 the amount of covalently bound isotope decreased to 42 +/- 11%. This is the first study to describe the long-term hepatic retention of microcystin toxin and documents putative detoxication products.
A Photodetoxification Mechanism of the Cyanobacterial Hepatotoxin Microcystin-LR by Ultraviolet Irradiation
摘要：
When microcystin-LR was exposed to UV, three major nontoxic compounds were formed. These compounds were identified as [4(E),6(Z)-Adda(5)] and [4(Z),6(E)-Adda(5)]microcystin-LR, which are geometrical isomers of the Adda [3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4(E),6(E)-decadienoic acid] moiety of microcystin-LR, and a novel compound, tricyclo-Adda [(2S,3S,1'R,3'S,4'S,5'R,6'R,7'R)-3-amino-5-(4',6'-dimethyl-3'-methoxytricyclo[5.4.0.0(1',5')]undeca-8',10'-dien-6'-yl)-2-methyl-4(E)-pentenoic acid]-containing microcystin-LR ([tricyclo-Adda(5)]microcystin-LR), which was formed by [2 + 2] addition between the benzene ring and the double bond at position 6-7 of the Adda moiety of the microcystin. The geometrical isomers were formed reversibly, and their equilibrium constants were almost the same. [Tricyclo-Adda(5)]microcystin-LR was also formed reversibly and was decomposed under UV light. These results suggest that the breakdown of microcystin-LR by UV irradiation proceeds via [tricyclo-Adda(5)]microcystin-LR.
A Photodetoxification Mechanism of the Cyanobacterial Hepatotoxin Microcystin-LR by Ultraviolet Irradiation
摘要：
When microcystin-LR was exposed to UV, three major nontoxic compounds were formed. These compounds were identified as [4(E),6(Z)-Adda(5)] and [4(Z),6(E)-Adda(5)]microcystin-LR, which are geometrical isomers of the Adda [3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4(E),6(E)-decadienoic acid] moiety of microcystin-LR, and a novel compound, tricyclo-Adda [(2S,3S,1'R,3'S,4'S,5'R,6'R,7'R)-3-amino-5-(4',6'-dimethyl-3'-methoxytricyclo[5.4.0.0(1',5')]undeca-8',10'-dien-6'-yl)-2-methyl-4(E)-pentenoic acid]-containing microcystin-LR ([tricyclo-Adda(5)]microcystin-LR), which was formed by [2 + 2] addition between the benzene ring and the double bond at position 6-7 of the Adda moiety of the microcystin. The geometrical isomers were formed reversibly, and their equilibrium constants were almost the same. [Tricyclo-Adda(5)]microcystin-LR was also formed reversibly and was decomposed under UV light. These results suggest that the breakdown of microcystin-LR by UV irradiation proceeds via [tricyclo-Adda(5)]microcystin-LR.
The band structure of multicomponent semiconductor photocatalysts, as well as their reactivity distinction under different wavelengths of light, is still unclear. BiOBr, which is a typical multicomponent semiconductor, may have two possible valence‐band structures, that is, two discrete valencebands constructed respectively from O 2p and Br 4p orbitals, or one valenceband derived from the hybridization
A hybrid adsorbent/photocatalyst was obtained and used for the removal of microcystin-LR, a potent toxin, from water via adsorption and photocatalyzed oxidation with singlet oxygen.
Mechanistic Study and the Influence of Oxygen on the Photosensitized Transformations of Microcystins (Cyanotoxins)
作者：Weihua Song、Sabrina Bardowell、Kevin E. O'Shea
DOI：10.1021/es063066o
日期：2007.8.1
Microcystins (MCs) produced by cyanobacteria are strong hepatotoxins and classified as possible carcinogens. MCs pose a considerable threat to consumers of tainted drinking and surface waters, but the photochemical fate of dissolved MCs in the environment has received limited attention. MCs are released into the environment upon cell lysis along with photoactive pigments including phycocyanin and chlorophyll a. The concentrations of MCs and pigments are expected to be greatest during a bloom event. These blooms occur in sunlit surface water and thus MCs can undergo a variety of solar initiated or photosensitized transformations. We report herein the role of oxygen, sensitizer, and light on the photochemical fate of MCs. The phycocyanin photosensitized transformation of MCs is elucidated, and photosensitized isomerization plays an important role in the process. The UV-A portion of sunlight was simulated using 350 nm light and the phototransformations of three MC variants (-LR, -RR, -LF) were investigated. Singlet oxygen leads to photooxidation of phycocyanin, the predominant pigment of cyanobacteria, hence, reducing the phototransformation rate of MCs. The phototransformation rate of MC-LR increases as pH decreases. The pH effect may be the result of MCs association with phycocyanin. Our results indicate photosensitized processes may play a key role in the photochemical transformation of MCs in the natural water.
Structures of three new cyclic heptapeptide hepatotoxins produced by the cyanobacterium (blue-green alga) Nostoc sp. strain 152
作者：Michio Namikoshi、Kenneth L. Rinehart、Ryuichi Sakai、Kaarina Sivonen、Wayne W. Carmichael
DOI：10.1021/jo00312a019
日期：1990.12
Mehrotra, Amit P.; Webster, Kerri L.; Gani, David, Journal of the Chemical Society. Perkin transactions I, 1997, # 17, p. 2495 - 2511
作者：Mehrotra, Amit P.、Webster, Kerri L.、Gani, David
