3D-structure of microcystin-LR
The chemical structure of microcystin-LR
|Jmol-3D images||Image 1|
|Molar mass||995.17 g mol−1|
|Solubility in ethanol||1 mg/mL|
|R-phrases||R26/27/28, R36/37/38, R43|
|S-phrases||S26, S36/37/39, S45|
| (what is: / ?)
Except where noted otherwise, data are given for materials in their standard state (at 25 °C (77 °F), 100 kPa)
- 1 History
- 2 Structure
- 3 Biosynthesis
- 4 Mechanism of toxicity
- 5 Indications
- 6 Available forms
- 7 Disposition and metabolism
- 8 Toxicity
- 9 References
The Chinese general Zhu Ge-Lin was the first to observe cyanobacteria poisoning about 1000 years ago. He reported the death of troops who drank green coloured water from a river in southern China. The first published report of an incidence of cyanobacteria poisoning dates from the poisoning of an Australian lake in 1878. Also, in China and Brasil, people died after drinking water from a lake. All these incidences have been attributed to cyanobacteria and the toxic compound microcystin-LR. That is the reason why the WHO, World Health Organisation, issued a guideline for microcystins in drinking water. The WHO guideline for microcystins in drinking water, based on microcystin-LR, is 1 μg/L.
Microcystins are cyclic heptapeptides. The seven peptides that are involved in the structure of a microcystin include a unique β-amino acid (Adda), which molecular formula is (2S,3S,8S,9S,4E,6E)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid. It contains D-alanine (D-ala) and D-methyl-aspartate (Masp). Furthermore, microcystins contain two variable residues, which make the differentiation between variants of microcystins. These two variable elements are always L-amino acids. In microcystin-LR these variable residues are L-arganine and L-leucine.
Until now over 80 microcystins are identified, representing differences in the two variable residues and some modifications in the other amino acids. These modifications include demethylation of Masp and Mdha and methylesterification of D-Glu. The different microcystins have different toxicity profiles, where microcystin-LR is found to be the most toxic one.
As microcystins are small polypeptides, they need not be synthesized at the ribosomes, they are in fact nonribosomal peptides. In Microcystis aeruginosa microcystin-LR is synthesized by proteins that are coded in a 55 kb microcystin-gene cluster (mcy) that contains 6 large (over 3 kb) genes that encode proteins with polyketide synthase activity as well as nonribosomal peptide synthase activity (mcyA-E and G) and 4 smaller genes (mcyF and H-J). These large proteins are made up of different protein domains, coined 'modules', that each have their own specific enzymatic function. Although the enzyme systems involved in the biosynthesis of microcystins is not identical among all cyanobacteria, there are large similarities and most of the essential enzymes are conserved.
The biosynthesis of microcystin-LR in Microcystis aeruginosa begins with the coupling of phenylacetate to the mcyG enzyme. In a series of reactions, catalysed by different enzymmodules as well as different enzymes, microcystin-LR is formed. The entire biosynthesis of microcystin-LR in Microcystis aeruginosa can be see in the figure.
The first steps of the synthesis involve the insertion of several carbon- and oxygenatoms between the acetyl- and phenylgroup. This part of the synthesis is catalyzed by enzymedomains that possess β-ketoacylsynthase, acyltransferase, C-methyltransferase and ketoacyl reductase activity. At the end of this stage, that is, after the first condensation of glutamate, the amino acid Adda is formed. The second part of the synthesis involves the condensation of the amino acids of which the microcystin is composed. Thus, in the case of microcystin-LR the consecutive condensation of the amino acids glutamic acid, methyldehydroalanine, alanine, leucine, methylaspartic acid and arginine leads to the coupled product. A nucleophilic attack of the nitrogen in the Adda residue results in the release of the cyclic microcystin-LR.
The different microcystins are all synthesized by the same enzymes as microcystin-LR.
Mechanism of toxicity
Microcystin-LR inhibits protein phosphatase type 1 and type 2A (PP1 and PP2A) activities in the cytoplasm of liver cells. This leads to an increase in phosphorylation of proteins in liver cells. The interaction of microcystin-LR to the phosphatases includes the formation of a covalent bond between a methylene group of microcystin-LR and a cystine residue at the catalytic subunit of the phosphoprotein phosphatase (PPP) family of serine/threonine-specific phosphatases, like PP1 and PP2A. When microcystin-LR binds directly to the catalytic center of the PPP enzymes, they block the access of the substrate to the active site completely and inhibition of the enzyme takes place. In this way the protein phosphatase is inhibited and more phosphorylated proteins in the liver cells are left, which is responsible for the hepatotoxicity of microcystin-LR.
The active site of catalytic PPP enzymes represents three surface grooves: the hydrophobic groove, the acidic groove and the C-terminal groove, which are Y-shaped with the active site at the bifurcation point. The Adda side-chain of microcystin-LR is accommodated to the hydrophobic groove, the carboxylic D-Glu site makes hydrogen bonds to metal-bound water molecules and the carboxyl group of the Masp site makes hydrogen bonds to conserved arginine and tyrosine residues in the PPP enzyme. Finally the methylene group at the Mdha site of microcystin-LR binds covalently to a S-atom of a cysteine residue, and the leucine residue packs closely to another conserved tyrosine residue.
Microcystin-LR is toxic for both humans and animals. There are epidemiological results from studies that have shown symptoms of poisoning attributed to the presence of cyanotoxins in drinking water. The effects are divided in short-term and long-term effects.
There are no reports known of human death by microcystin-LR, although there are reports of health effects after exposure. One of the most outstanding reports, is an outbreak in Brazil. 116 patients experience multiple effects: visual disturbance, nausea, vomiting and muscle weakness. One hundred developed acute liver failure and 52 suffered from symptoms of what is called “Caruaca Syndrome”. This syndrome was caused by dialysis with water, that had not been treated.
There are a few effect administered for exposure to microcystin-LR. Microcystin is in general a liver toxin. Most of the toxicity studies have been done with mice, that received intra-peritoneal injections. The most common effect is liver damage  Two of the mostly seen symptoms are Gastroenteritis and cholestatic liver disease. Microcystin-LR is a hepatotoxic compound.
A few results from mice experiments were as follows. The mice died within a few hours from the injection, with a lethal dose of micocystin-LR. The liver damage is noticeable in 20 minutes. Within a few hours, the liver cells die.
Short-term effects may result in long-term injury and chronic low-level exposure may cause adverse health effects. From animal studies, it is proven that there will be chronic liver injury from oral exposure to microcystin-LR. It even might be carcinogen. Cancers were found during animal studies. Microcystin-LR itself does not cause cancer, but it may stimulate the growth of cancer cells.
Microcystin-LR had effects on all animals, not only the domestic animals from swimming in a river of drinking water with cyanobacteria blooms. Symptoms in domestic animal poisoning include diarrhea, vomiting, weakness, recumbency and are fatal in most cases
Mircocystin-LR is toxic for all animals, including the animals we consume as humans. Fishes and birds are also at risk for microcystin-LR poisoning.
Cyanobacteria prefer to live in water bodies such as lake, ponds, reservoirs, and slow-moving streams. When the water is warm there are enough nutrients available for the bacteria to survive. The most cyanobacteria produce a group of toxins, of which microcystin is one. When a cyanobacterium dies, its cell wall degrades and the toxins are released in the water. Microcystins are extremey stable in water and withstand chemical breakdown such as hydrolysis or oxidation. The half-life of this toxin is 3 weeks at pH 1 and 40°C. At typical conditions in the environment, however, the half-life is 10 weeks.
After release in the water, microcystins are actively absorbed by fish and birds from intoxicated water and thus enter the food chain. Humans are also exposed to microcystins by performing activities in intoxicated water.
Disposition and metabolism
Microcystin-LR is rapidly excreted from the blood plasma. Plasma half-lifes for the α- and β-stages, corresponding to distribution and elimination, are respectively 0.8 and 6.9 minutes. The total clearance of the compound from the plasma is about 0.9 mL/min. The excretion of the compound takes primarily place via the feces and urine. After 6 days approximately 24% of the intake is excreted from the body, of which about 9% is excreted via the feces and 14.5% via the urine.
Microcystin-LR is mostly concentrated in the liver. Other tissues get exposed at much lower levels.
Data about the metabolism of microcystin-LR in humans is very scarce. Data about metabolism and disposition of the toxin in mice and rats is more widely available. In these animals microcystin-LR is rapidly concentrated in the liver. Intoxication of mice with microcystin-LR led to a decrease in the levels of cytochrome P450 and cytochrome b5 and an increase in cytochrome P420, to which CYP450 is converted. Together with the fact that mice with an induced higher concentration CYP450 are less affected by the toxin, this suggest that CYP450 plays an important role in the detoxification of the compound.
In phase 2 of the biotransformation the compound is conjugated with several different endogenous substances. Microcystin-LR is known to be excreted as glutathione conjugate, cysteine conjugate and an oxidized ADDA diene conjugate. The glutathione and cysteine conjugate with the Mda-moiety. The oxidized ADDA is conjugated at the conjugated bond.
Toxicity of cyanotoxins is very diverse and include neurotoxicity, hepatotoxicity, cytotoxicity and dermatotoxicity. Microcystins are generally associated with hepatotoxicity. The toxic effect of microcystins is due to their inhibition of protein phosphatases.
Acute subacute toxicity
Lots of studies took place with intraperitoneal administration. Because of the differences in lipophilicity and polarity between the different microcystins, it cannot be presumend that the i.p. LD50 will predict toxicity after oral administration.
Microcystins are hepatotxins. After acute exposure, severe liver damage is noticeable by a disruption of liver cell structure. The liver weight will increase due to intrahepatic hemorrhage, haemodynamic shock, heart failure and death.
After nasal administration of microcystin-LR, the epithelium of nasal mucosa of both the olfactory and respiratory zones were suffering from necrosis. Even liver lesions were noticed after oral administration. The LD50 for nasal administration is equal to the intraperitoneal administration.
Repeated oral administration
For the assessment of possible chronic human health effects, studies involving repeated oral administration of pure microcystins at various dose levels are most desirable. In a mice study, pure mirocystin-LR was administered orally at doses 0, 40, 200 or 1000 μg/kg bodyweight. At the highest dose, almost all mice showed liver changes and chronic inflammation and a few other symptoms. In female mice only changes in transaminases were observed at the highest dose.
Mice showed neoplastic liver nodules after 100 oral administrations at 20 μg/kg bodyweight. The nodules observed were up to 5mm in diameter. However, no mice showed liver nodules after 100 administrations of 80 μg/kg.
Interaction with tumors
The IARC committee concluded that microcystin-LR is possibly carcinogenic to humans. So, microcystin-LR itself is not a carcinogen, but it stimulates tumor growth. Mice treated with the carcinogenic compound dimethylbenzathracene showed an increased number and weight of skin tumors.
In vivo animal experiments
There is very little known about acute toxicity for humans, but there have been animal studies, showing the following results.
|Method of administration ||Toxicity||Species||Value|
|Inhalation, 10h||LC50||Mouse||18 mg/kg|
When microcystins are injected intraveneously or intraperitoneally, they localize in the liver. This appears to be the result of uptake by hepatocytes. The WHO repport states that microcystins are lethal to mice when they are exposed intraperitoneally to 25 to 150 µg/kg body weight. Perhaps due to poor absorption after exposure, orally administered microscytins are less toxic, as a lethal dose in mice is about 5 to 10 µg/kg body weight. Hepatotoxicity in the form of hepatic necrosis occurs within 60 minutes after an intraveneous dose. Blooms of Microcystis aeruginosa did not cause increased tumor rates in groups of mice treated for up to one year. It is shown that mice given 20 µg/kg body weight 4 times a week during a period of 28 weeks developed neoplasms of the liver. There results are, however, ambiguous. By the oral route, microcystin-LR displays acute toxicity in rodents. It is apparent that a significant amount of the oral dose passes the intestinal barrier.
Microcystins do not appear to show developmental toxicity.
The WHO states microcystin-LR has no mutagenic effect. However, the induction of DNA strand-breaks in lymphocytes has been observed in mice after single oral administration. The effect is time- and dose-dependent. There is no change in the expression of selected genes involved in the cellular response to DNA damage after a 4 hour exposure. After 24 hours, the DNA damage-responsive genes were upregulated, which indicates that microcystin-LR is an indirect genotoxic agent. In China, the highest incidence of liver cancer occurs in areas with abundant cyanobacteria in the surface waters. Tumor development is associated with low-concentration exposure over a long period of time.
In vitro studies
In vitro studies showed that microcystin-LR is a potent inhibitor of protein phosphatases 1 (PP-1) and PP2A, but has no effect on protein kinase C or cyclic AMP-dependent kinase. Mutagenicity does not appear to occur for purified toxins derived from Microcystis, although the toxins were clastogenic for human lymphocytes.
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