Nodularins are potent toxins produced by the cyanobacterium Nodularia spumigena, among others. This aquatic, photosynthetic cyanobacterium forms visible colonies that present as algal blooms in brackish water bodies throughout the world. The late summer blooms of Nodularia spumigena are among the largest cyanobacterial mass occurrences in the world. Cyanobacteria synthesize many toxic substances, most notably microcystins and nodularins: the two are not easily differentiated. A significant homology of structure and function exists between the two, and microcystins have been studied in greater detail. Because of this, facts from microcystins are often extended to nodularins. Nodularin-R is the predominant toxin variant, though 10 variants of nodularin have been discovered to date. Nodularins are cyclic nonribosomal pentapeptides and contain several unusual non-proteinogenic amino acids such as N-methyl-didehydroaminobutyric acid and the β-amino acid ADDA. These compounds are relatively stable compounds: light, temperature, and microwaves do little to degrade the compounds. Nodularins are often attributed to gastroenteritis, allergic irritation reactions, and liver diseases. Nodularin-R is most notorious as a potent hepatotoxin that may cause serious damage to the liver of humans and other animals. The WHO drinking water concentration limit for nodularins (extended from microcystins-LR) is 1.5 ug /L.
Physiochemical properties Nodularin-R has a molecular formula C41H60N8O10 and average molecular weight of 824.963 g/mol. The compound has 8 defined stereocenters. It is a solid substance. In methanol, nodularin is soluble 2 mg/mL. It breaks down slowly at temperatures greater than 104 °F (40 °C), pH less than 1 and pH greater than 9. Nodularins are typically resistant to breakdown via hydrolysis and oxidation in aquatic conditions. Hazardous decomposition products of nodularins are carbon monoxide and carbon dioxide.
The basic framework for nodularin structure is D-Masp1-Z2-Adda3-D-γ-Glu4-Mdhb5, where Z is a variable amino acid; the systematic name "nodularin-Z" (NOD-Z in short) is then assigned based on the one letter code (if available; longer code otherwise) of the amino acid. For the common NOD-R, the Z amino acid is arginine.
Mechanism of action
Metabolism Nodularin primarily targets the liver, though nodularins also accumulate in the blood, intestines, and kidneys. In the liver, this targeting leads to cytoskeletal damage, necrosis, and rapid blistering of the hepatocytes. Cell death and rapid blistering also destroys the finer blood vessels of the liver. The damage results in blood pooling in the liver, which can lead to an increase in liver weight of 100%. Death by nodularin poisoning occurs from this hemorrhagic shock. This is fast acting, and occurs within a few hours after a high dose. At the molecular level and in further detail, nodularin is processed in a complex manner to induce toxic effects. During digestion, nodularins diffuse from small intestine into liver due to active uptake by an unspecific organic anion transporter in the bile acid carrier transport system. This transporter is expressed in the gastrointestinal tract, kidney, brain, and liver. Once in the liver, nodularin inhibits three key enzymes, specifically the catalytic units of serine/threonine protein phosphatases: protein phosphatase 1 (PP-1) and protein phosphatase 2A. (PP-2A), and protein phosphatase 3 (PP-3). These enzymes act by removing the phosphate from a protein, inhibiting the function of the protein.
An initial noncovalent interaction involving the ADDA side chain (specifically where ADDA has a 6E double bond) of the nodularin and a free D-glutamyl carboxyl group off a cyclic structure of the phosphatase is the source of toxicity. The ADDA group blocks enzyme (phosphatase) activity by interacting with hydrophobic groove and obstructing substrate access to active site cleft. The toxin-phosphatase bond interactions (nodularin-PP-1, nodularin-PP-2A) are extremely strong. This leads to inhibition of the enzyme activity. Of note, nodularins differ from microcystins here: nodularins bind noncovalently to protein phosphatases while microcystins bind covalently. A further interaction involves a Michael-addition covalent linkage of electrophilic α, β unsaturated carbonyl of a methyldehydroalanine residue on the nodularin to a thiol of cysteine 273 on PP-1. Though the covalent bond in step 2 is not essential for inhibition of enzyme activity, it does help mediate activity. Without this covalent bond, there is over a 10-fold reduction of nodularin affinity for the phosphatase. The inhibition of the protein phosphatases results in increased phosphorylation of cytoskeletal proteins and cytoskeletal associated proteins. The hyperphosphorylation of intermediate filaments of the cell, specifically of cytokeratin 8 and cytokeratin 18, is the main cause for protein imbalance. The protein imbalance stimulates redistribution and rearrangement of these proteins, which changes the whole cell morphology and membrane integrity. More specifically, this redistribution leads to collapse of actin microfilaments in the hepatocyte cytoskeleton and dislocation of a-actinin and talin. Contact with neighboring cells is reduced and sinusoidal capillaries lose stability which rapidly leads to intrahepatic hemorrhage and often results in serious liver malfunction or death.
Reactive oxidative species Nodularins are further implicated in the formation of reactive oxidative species (ROS), specifically superoxide and hydroxyl radicals, which consequently cause oxidative DNA damage via peroxidation of lipids, proteins, and DNA via an unknown mechanism.
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