Microcystin-LR (MC-LR) is a toxin produced by cyanobacteria. It is the most toxic of the microcystins.
Structure Microcystins are cyclic heptapeptides. The seven amino acids that are involved in the structure of a microcystin include the unique amino acids ADDA and D-β-methyl-isoaspartate (D-β-Me-isoAsp). Furthermore, microcystins contain two variable residues, which make the differentiation between variants of microcystins. These two variable functionalities are always standard proteinogenic amino acids - In microcystin-LR these are leucine and arginine.
More than 250 microcystins have been identified to date,Structural Diversity, Characterization and Toxicology of Microcystins, doi: 10.3390/toxins11120714 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. Different microcystins have different toxicity profiles, with microcystin-LR found to be the most toxic.S. Pereira, V. Vasconcelos & A. Antunes, Computational study of the covalent bonding of microcystins to cysteine residues - a reaction involved in the inhibition of the PPP family of protein phosphatases, FEBS Journal, doi: 10.1111/j.1742-4658.2011.08454.xA. Campos & V. Vasconcelos, Molecular Mechanisms of Microcystin Toxicity in Animal Cells, Int. J. Mol. Sc., 11(1), pp. 268-287
Biosynthesis Microcystins are small nonribosomal peptides. In Microcystis aeruginosa microcystin-LR is synthesized by proteins that encoded by a 55 kb microcystin-gene cluster (mcy) that contains 6 large (over 3 kb) genes that encode proteins with polyketide synthase activity, 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.D. Tillett et al., Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system, Chem. Biol., 7(10), pp. 753-764 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.G. Christiansen et al., Microcystin Biosynthesis in Planktothrix: Genes, Evolution, and Manipulation, J. Bacteriol., 185(2), pp. 564-572
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 enzyme modules as well as different enzymes, microcystin-LR is formed. The entire biosynthesis pathway of microcystin-LR in Microcystis aeruginosa is illustrated in the figure.
The first steps of the synthesis involve the insertion of several carbon- and oxygen atoms between the acetyl- and phenylgroup. This part of the synthesis is catalyzed by enzyme domains 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.T. Nishizawa et al., Polyketide Synthase Gene Coupled to the Peptide Synthetase Module Involved in the Biosynthesis of the Cyclic Heptapeptide Microcystin, J. Biochem., 127(5), pp.779-789
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.
Effects 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.
Human poisonings There are no verifiable reports of human deaths known to have been specifically caused by microcystin-LR, although there are reports of health effects after exposure and there have been deaths attributed to microcystins in general.Bulter, N., Carlisle, J.C. Microcystins: A brief overview of their toxicity and effects, with special reference to fish, wildlife and livestock. Department of Water Resources, California. January 2009. One of the most outstanding reports was an outbreak in Caruaru, Brazil, in 1996. 116 patients experienced multiple effects: visual disturbance, nausea, vomiting and muscle weakness. One hundred developed acute liver failure and 52 suffered from symptoms of what is now called "Caruaru Syndrome."Azevedo, S.M. et al., Human intoxication by microcystins during renal dialysis treatment in Caruaru-Brazil. Toxicology, 2002. 181-182: p. 441-6. The syndrome was caused by dialysis therapy with water that had not been properly treated.Jochimsen, E.M. et al., Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. N Engl J Med, 1998. 338(13): p. 873-8.
Short-term effects There are few short-term effects caused by exposure to microcystin-LR. Microcystins are primarily hepatotoxic compounds; therefore, noticeable toxic effects are not immediate. Most of the toxicity studies have been done with mice that received intra-peritoneal injections. The most common effect is liver damage,WHO (2003) Cyanobacterial toxins: Microcystin-LR in drinking-water. Background document for preparation of WHO Guidelines for drinking-water quality. Geneva, World Health Organization WHO/SDE/WSH/03.04/57). Two of the most commonly seen symptoms are gastroenteritis and cholestatic liver disease.
In an experiment with mice, the animals died within a few hours after injection of a lethal dose of micocystin-LR. Liver damage could be noticed in 20 minutes. Within a few hours, liver cells died.Slatkin, D.N., et al., Atypical pulmonary thrombosis caused by a toxic cyanobacterial peptide. Science, 1983. 220(4604): p. 1383-5.
Long-term effects Acute microcystin-LR intoxication may result in long-term injury, while 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 might even be carcinogenic. Cancers have been found during animal studies. Microcystin-LR itself does not cause cancer, but it may stimulate the growth of cancer cells.
Animal effects 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 casesDeVries, S.E., et al., Clinical and pathologic findings of blue-green algae (Microcystis aeruginosa) intoxication in a dog. Journal of Veterinary Diagnostic Investigation, 1993. 5(3): p. 403.Briand, J.F., et al., Health hazards for terrestrial vertebrates from toxic cyanobacteria in surface water ecosystems. Vet Res, 2003. 34(4): p. 361-77.
Mircocystin-LR is toxic for all animals, including the animals consumed by humans. Fishes and birds are also at risk for microcystin-LR poisoning.
Exposure Routes 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. Most cyanobacteria produce toxins, of which microcystin is only one group. When a cyanobacterium dies, its cell wall degrades while the toxins are released in the water. Microcystins are extremely 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. Microcystin-LR water contamination is resistant to boiling and microwave treatments.
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.Harada, K.I., et al., Stability of microcystins from cyanobacteria. III. Effect of pH and temperature Phycologia, 1996. 35(6) pp. 83-88
Disposition and metabolism
Disposition Microcystin-LR is rapidly excreted from the blood plasma. Plasma half-lives for the α- and β-stages, corresponding to distribution and elimination, are respectively 0.8 and 6.9 minutes.Robinson, N.A., Pace, J.G., Matson, C.F., Miura, G.A. and Lawrence, W.B. 1991 Tissue distribution, excretion and hepatic biotransformation of microcystin-LR in mice, J.Pharmacol. Exp. Ther., 256(1), 176-182. 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.
Metabolism 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.Brooks, W.P. and Codd, G.A. 1987 Distribution of Microcystis aeruginosa peptide toxin and interactions with hepatic microsomes in mice Pharmacol. Toxicol., 60(3), 187-191. 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.