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Orellanine or orellanin is a mycotoxin found in a group of mushrooms known as the Orellani of the family Cortinariaceae. Structurally, it is a bipyridine N-oxide compound somewhat related to the herbicide diquat.

History
Orellanine first came to people's attention in 1957 when there was a mass poisoning of 135 people in Bydgoszcz, Poland, which resulted in 19 deaths. Orellanine comes from a class of mushrooms that fall under the genus Cortinarius. Although not all the species in this genus are poisonous/contain orellanine, it was found that Cortinarius orellanus, rubellus, henrici, rainerensis and bruneofulvus contain orellanine. Poisonings pertaining to these mushrooms were predominately in Europe where foraging was a major source of nourishment, although there are cases of orellanine poisoning in North America as well. Orellanine has been found to cause acute kidney injury and there are many cases where people have taken this mushroom mistaking it for causing hallucinogenic effects.

It wasn't until 1962 until the first isolation of orellanine was done. The first methanolic extraction and isolation of orellanine was done by Stanisław Grzymala and isolated from the mushroom Cortinarius orellanus. Along with the isolation of orellanine, Grzymala was also able to demonstrate the nephrotoxicity of Cortinarius orellanus and determine various physical and chemical properties of orellanine. He found that the toxicity of the mushrooms was linked to delayed and acute kidney injury, as well as when the isolated white crystalline substance was heated above 150 ̊C it began to slowly decompose. After this first isolation of orellanine, the structure of orellanine was first discovered by Antkowiak and Gessner in 1979. Orellanine’s structure was found to be 3,3',4,4'-tetrahydroxy- 2,2'-bipyridine-l,l'-dioxide. Antkowiak and Gessner were also able to determine that orellanine was the mono-N-oxide of orelline, which was the decomposition product of orellanine. It was also found that orelline was non-toxic. The first successful synthesis of orellanine was done in 1985. Tiecco, M. et. Al. completed a total synthesis of orellanine using commercially available 3-hydroxypyridine. After the first successful synthesis, the structure was confirmed in 1987 by Cohen-Addad et al. in 1987 by X-ray crystallography.

Synthesis
The chemical constitution of orellanine remained unknown until the Polish chemists Antkowiak and Gessner in the last half of the 1970s discovered that it was a bipyridine dioxide. Orellanine undergoes tautomerization, and the more stable tautomer is the amine oxide form. An interesting feature of orellanine is its ability to bind aluminium ions to form chelation complexes.



The first synthesis of orellanine was done in 1985 and Tiecco, M. et. Al. were able to synthesize orellaine from 3-hydroxy pyridine. This synthesis was completed in 8 steps with a 79-87% yield. When synthesized the two pyridyl rings are nearly perpendicular and the molecule is chiral. When it is isolated from the mushroom, it is an optically inactive racemic mixture. Other synthetic strategies have also been attempted. For example, orellanine was also synthesized in 9 steps by Dehmlow and Schulz in 1985 using 3-aminopyridine and the desired product was synthesized with 30% yield.

In the synthesis done by Tiecco, M. et al. can be seen in the scheme below. In the first step, 3-hydroxy pyridine was first treated with bromine in an alkaline solution to obtain 2. The product of that step was then subjected to O-alkylation using DMF as a solvent to obtain 3. 3 was the oxidized with m-chloroperbenzoic acid in chloroform to give 4. That product was then nitrated with nitric acid and sulfuric acid to obtain a mixture of 5 and 6. These two molecules were separated by combining the mixture of products with water. 5 is insoluble in water, whereas 6 is soluble in water. 6 was then subjected to sodium methoxide in methanol to obtain the other methoxy group seen in product 7 and 7 was deoxygenated using phosphorus tribromide to obtain 8. To obtain tetramethyl orelline, structure 9, triphenylphosphine, NiCl2·6H2O, and zinc powder were used to conduct the homocoupling of halopyridines through the use of nickel-phosphine complexes. Once 8 with the bipyridyl structure had been obtained, the synthesis of orellanine could be conducted. The bipyridyl product from number 8 is then dealkylated with hydrobromic acid to give orelline, which was found to be a yellow crystalline solid (10). Orelline is then oxidized with hydrogen peroxide using heat to obtain the desired product, orellanine (11).



Another way to synthesize orellanine from 9 was to subject it to excess m-chloro perbenzoic acid in chloroform to obtain 12, tetramethyl orellanine. Tetramethyl orellaine was then demethylated using hydrobromic acid to yield orellanine.



Biosynthesis
Orellanine occurs in nature in mushrooms of the family Cortinariacae. The biosynthetic pathway of orellanine is a subject of ongoing investigation, and has not been fully illucidated. The most recent studies performed on its biosynthesis were able to identify anthranilic acid as a precursor to orellanine. In a feeding study, radiolabeled anthranilic acid was injected into the fruiting bodies of the mushrooms. Later, the fruiting bodies were collected and the orellanine was isolated from them. It was found that the orellanine was enriched in N15, conclusively identifying anthranilic acid as a biosynthetic precursor to orellanine. However, no further additional biosynthetic steps have been definitively identified. In a similar experiment, radiolabeled glucose and amino acids were injected into the mushrooms, and no significant enrichment in N15 or C13 was observed in orellanine, indicating that orellanine is not synthesized from these precursors. During the course of this study, it was also determined that the direct precursor to orellanine is orellanine diglucoside. A complete biosynthetic pathway (see below) was proposed as a part of this study, but has yet to be experimentally verified.



Chemical Properties
In its pure form, orellanine is a fine colorless crystal that fluoresces a navy blue color under ultraviolet irradiation. At first, the fluorescence from orellanine is a dark blue color, but progresses to a bright turquoise due to a photochemical reaction. In crystalline form, the planar pyridine rings are oriented perpendicular to each other, effectively making orellanine chiral when in crystalline form. However, any orellanine isolated from nature is racemic, owing to the low energetic barrier to rotation of the central bond. In solution, orellanine exists as two tautomers, with the more stable being the amine oxide form. Orellanine is generally very thermally stable and does not decompose at cooking temperatures, but at 267C, orellanine will decompose explosively. Over time, due to irradiation, orellanine will decay into the less toxic orelline by the release of oxygen.

Chemical Mechanisms of Toxicity
Orellanine is a member of a family of compounds known as bipyridines, consisting of two mono-nitrogen substituted 6 membered aromatic rings. The signature reaction that these molecules are best known for is the catalysis of the production of reactive oxygen species (ROS) in biological systems. The exact mechanism by which this occurs in orellanine is the subject of ongoing study, but it in chemically similar molecules such as paraquat, the reaction is known to involve single electron transfer mechanisms that consume biological electron donors such as NADPH to produce O2-, or superoxide.

The first experiments to study orellanine's toxicity were performed by simply adding the toxin to mammalian kidney cell culture. In this system, it was observed that a diverse array of macromolecular synthesis processes were inhibited by treatment with orellanine. DNA, RNA, and protein synthesis were each inhibited by 85%, 80%, and 73% respectively in live cells. In in-vitro protein synthesis systems, no inhibition of protein synthesis occurred, indicating that orellanine does not interact directly with the ribosome or translational machinery. Rather, it was concluded that a metabolite of orellanine inhibits protein synthesis.

One of the first discoveries regarding orellanine's chemical mechanism of toxicity was that the active form of orellanine that is an ortho-semiquinone, indicating that the toxin becomes reactive by adopting a free-radical electronic structure. The general reaction scheme proceeds by a series of single electron transfer events. Two mechanisms have been shown to oxidize orellanine to its reactive semiquinone form. The first is a photochemical oxidation, and the second uses Fe2 as an electron source to initiate oxidation of orellanine. The iron-based activation mechanism is biologically relevant due to the prevalence of heme-containing enzymes that occur in cells. This electron is then subsequently transferred from the semiquinone orellanine to a molecular oxygen, producing superoxide. Superoxide then subsequently decays into other reactive oxygen species.



Under the proposed model in which orellanine acts as a catalyst for the production of ROS, the toxic effects of Orellanine don’t occur due to the direct action of the orellanine molecule itself, but due to the reactivity of the oxy radicals that it produces. The systemic accumulation of ROS is known as oxidative stress and has diverse detrimental effects on cellular physiology. Under another series of experiments, orellanine has been shown to induce scission and double strand breaks in DNA. In an experiment using free iron as an electron source, orellanine was shown to induce DNA strand scission as a result of the production of oxy radicals. It was also concluded as a result of this study that orellanine comes into close contact with DNA because oxy radicals have a short lifetime and could not have been distally generated and then diffused before reacting with the DNA.

Clinical Symptoms and Treatment
The initial symptoms of orellanine poisoning usually consist of mild and nonspecific gastrointestinal symptoms, which does not usually prompt the seeking of medical attention from the patient. Orellanine has a long incubation time from the time of ingestion to the presentation of the first serious symptoms that may range from a few days to a few weeks, depending on the amount of the substance that is ingested. Due to this long incubation time, patients often do not associate the mushrooms that they ate with the symptoms that they experience. The nonspecific gastrointestinal symptoms progress to renal oriented symptoms including but not limited to lower back pain, strong thirst, and oligouria. The symptoms can progress to acute renal failure depending on the case and the severity of the poisoning.

Clinical samples of urine and blood do not typically contain detectable amounts of orellanine because, due to the long incubation time, the poison is not readily detected in urine or blood by the time that symptoms manifest. Because of this, orellanine poisoning is often misdiagnosed. Relatively little pharmacokinetic information is available on orellanine in humans, but in animal models, it has been shown that it is only excreted into urine for 24 hours after ingestion. However, the compound remains detectable in kidney tissue for months after ingestion, indicating that the compound is sequestered in the kidney in a form that does not easily exchange with the environment.

There are no known antidotes to orellanine poisoning, but there are a few reported strategies for treatment that have yielded various amounts of success. Because orellanine produces reactive oxygen species that cause tissue damage and renal failure, one clinically reported treatment was the antioxidant N-acetyl cysteine. The merit of this treatment is inconclusive even though the patient recovered because the case of poisoning was relatively mild and the patient would have likely recovered without treatment. Other standard treatments address acute renal failure and support renal function by hemodialysis.

Of patients that present with orellanine poisoning, only 30 percent fully regain kidney function, 20-40 percent require long term renal replacement therapy, and the majority recover partial renal function with some degree of permanent fibrosis.