Anatoxin-a: Difference between revisions

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| IUPACName=1-(9-azabicyclo[4.2.1]non-2-en-2-yl)ethan-1-one

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'''Anatoxin-a''', also known as '''Very Fast Death Factor''' ('''VFDF'''), is a secondary, bicyclic [[amine]] [[alkaloid]] and [[cyanotoxin]] with acute [[neurotoxicity]]. It was first discovered in the early 1960s in Canada, and was isolated in 1972. The toxin is produced by multiple genera of [[cyanobacteria]] and has been reported in North America, South America, Central America, Europe, Africa, Asia, and Oceania. Symptoms of anatoxin-a toxicity include [[Ataxia|loss of coordination]], [[Fasciculation|muscular fasciculations]], [[convulsion]]s and death by [[respiratory paralysis]]. Its [[mode of action]] is through the [[nicotinic acetylcholine receptor]] (nAchR) where it mimics the binding of the receptor's natural [[ligand]], [[acetylcholine]]. As such, anatoxin-a has been used for medicinal purposes to investigate diseases characterized by low acetylcholine levels. Due to its high toxicity and potential presence in drinking water, anatoxin-a poses a threat to animals, including humans. While methods for detection and water treatment exist, scientists have called for more research to improve reliability and efficacy. Anatoxin-a is not to be confused with [[guanitoxin]] (formerly anatoxin-a(S)), another potent cyanotoxin that has a similar mechanism of action to that of anatoxin-a and is produced by many of the same cyanobacteria genera, but is structurally unrelated.<ref name="Aráoz">{{cite journal | vauthors = Aráoz R, Molgó J, Tandeau de Marsac N | title = Neurotoxic cyanobacterial toxins | journal = Toxicon | volume = 56 | issue = 5 | pages = 813–28 | date = October 2010 | pmid = 19660486 | doi = 10.1016/j.toxicon.2009.07.036 }}</ref>

==History==

Anatoxin-a was first discovered by P.R. Gorham in the early 1960s, after several herds of cattle died as a result of drinking water from [[Saskatchewan Lake]] in Ontario, Canada, which contained toxic [[algal blooms]]. It was isolated in 1972 by J.P. Devlin from the cyanobacteria ''[[Anabaena flos-aquae]]''.<ref name="Botana">{{cite book | vauthors = Botana LM, James K, Crowley J, Duphard J, Lehane M, Furey A | chapter = Anatoxin-a and Analogues: Discovery, Distribution, and Toxicology. | title = Phycotoxins: Chemistry and Biochemistry | date = March 2007 | pages = 141–58 | publisher = Blackwell Publishing | doi = 10.1002/9780470277874.ch8 | isbn = 978-0-470-27787-4 }}</ref>

== Occurrence ==

Anatoxin-a is a neurotoxin produced by multiple genera of freshwater cyanobacteria that are found in water bodies globally.<ref name=":0"/> Some freshwater cyanobacteria are known to be salt tolerant and thus it is possible for anatoxin-a to be found in estuarine or other saline environments.<ref>{{cite web | date=June 2015|title=Health Effects Support Document for the Cyanobacterial Toxin Anatoxin-A|url=https://www.epa.gov/sites/production/files/2017-06/documents/anatoxin-a-report-2015.pdf|access-date=October 25, 2020|website=United States Environmental Protection Agency}}</ref> Blooms of cyanobacteria that produce anatoxin-a among other cyanotoxins are increasing in frequency due to increasing temperatures, [[Stratification (water)|stratification]], and [[eutrophication]] due to nutrient runoff.<ref>{{cite journal | vauthors = Paerl HW, Otten TG | title = Harmful cyanobacterial blooms: causes, consequences, and controls | journal = Microbial Ecology | volume = 65 | issue = 4 | pages = 995–1010 | date = May 2013 | pmid = 23314096 | doi = 10.1007/s00248-012-0159-y | bibcode = 2013MicEc..65..995P | s2cid = 5718333 }}</ref> These expansive cyanobacterial [[harmful algal bloom]]s, known as cyanoHABs, increase the amount of cyanotoxins in the surrounding water, threatening the health of both aquatic and terrestrial organisms.<ref>{{cite journal | vauthors = Miller TR, Beversdorf LJ, Weirich CA, Bartlett SL | title = Cyanobacterial Toxins of the Laurentian Great Lakes, Their Toxicological Effects, and Numerical Limits in Drinking Water | journal = Marine Drugs | volume = 15 | issue = 6 | page = 160 | date = June 2017 | pmid = 28574457 | pmc = 5484110 | doi = 10.3390/md15060160 | doi-access = free }}</ref> Some species of cyanobacteria that produce anatoxin-a don't produce surface water blooms but instead form [[Benthic zone|benthic]] mats. Many cases of anatoxin-a related animal deaths have occurred due to ingestion of detached benthic cyanobacterial mats that have washed ashore.<ref>{{cite web |date=November 2019|title=Cyanobacterial toxins: Anatoxin-a|url=https://www.who.int/water_sanitation_health/water-quality/guidelines/chemicals/anatoxin-a-gdwq-bd-for-review-20191122.pdf|access-date=October 25, 2020|website=World Health Organization}}</ref>

Anatoxin-a producing cyanobacteria have also been found in soils and aquatic plants. Anatoxin-a sorbs well to negatively charged sites in clay-like, organic-rich soils and weakly to sandy soils. One study found both bound and free anatoxin-a in 38% of aquatic plants sampled across 12 Nebraskan reservoirs, with much higher incidence of bound anatoxin-a than free.<ref>{{cite journal | vauthors = Al-Sammak MA, Hoagland KD, Cassada D, Snow DD | title = Co-occurrence of the cyanotoxins BMAA, DABA and anatoxin-a in Nebraska reservoirs, fish, and aquatic plants | journal = Toxins | volume = 6 | issue = 2 | pages = 488–508 | date = January 2014 | pmid = 24476710 | pmc = 3942747 | doi = 10.3390/toxins6020488 | doi-access = free }}</ref>

== Experimental studies ==

In 1977, Carmichael, Gorham, and Biggs experimented with anatoxin-a. They introduced toxic cultures of ''A. flos-aquae'' into the stomachs of two young male calves, and observed that muscular fasciculations and loss of coordination occurred in a matter of minutes, while death due to respiratory failure occurred anywhere between several minutes and a few hours. They also established that extensive periods of [[artificial respiration]] did not allow for detoxification to occur and natural neuromuscular functioning to resume. From these experiments, they calculated that the oral minimum lethal dose (MLD) (of the algae, not the anatoxin molecule), for calves is roughly 420&nbsp;mg/kg body weight.<ref>{{cite journal | vauthors = Carmichael WW, Gorham PR, Biggs DF | title = Two laboratory case studies on the oral toxicity to calves of the freshwater cyanophyte (blue-green alga) Anabaena flos-aquae NRC-44-1 | journal = The Canadian Veterinary Journal | volume = 18 | issue = 3 | pages = 71–5 | date = March 1977 | pmid = 404019 | pmc = 1697489 }}</ref>

In the same year, Devlin and colleagues discovered the bicyclic secondary amine structure of anatoxin-a. They also performed experiments similar to those of Carmichael et al. on mice. They found that anatoxin-a kills mice 2–5 minutes after [[intraperitoneal injection]] preceded by twitching, muscle spasms, paralysis and respiratory arrest, hence the name Very Fast Death Factor.<ref>{{cite journal| vauthors = Devlin JP, Edwards OE, Gorham PR, Hunter NR, Pike RK, Stavric B |date=2011-02-04|title=Anatoxin-a, a toxic alkaloid from Anabaena flos-aquae NRC-44h |journal=Canadian Journal of Chemistry |volume=55|issue=8|pages=1367–1371|doi=10.1139/v77-189|doi-access=free}}</ref> They determined the [[LD50]] for mice to be 250&nbsp;μg/kg body weight.<ref name="Aráoz" />

Electrophysiological experiments done by Spivak et al. (1980) on frogs showed that anatoxin-a is a potent agonist of the muscle-type (α₁)₂βγδ nAChR. Anatoxin-a induced depolarizing neuromuscular blockade, contracture of the frog's rectus abdominis muscle, depolarization of the frog sartorius muscle, desensitization, and alteration of the action potential. Later, Thomas et al., (1993) through his work with chicken α₄β₂ nAChR subunits expressed on mouse M 10 cells and chicken α₇ nAChR expressed in oocytes from ''[[Xenopus laevis]]'', showed that anatoxin-a is also a potent agonist of neuronal nAChR.<ref name="Aráoz" />

== Toxicity ==

=== Effects ===

Laboratory studies using mice showed that characteristic effects of acute anatoxin-a poisoning via [[intraperitoneal injection]] include [[Fasciculation|muscle fasciculations]], tremors, staggering, gasping, respiratory paralysis, and death within minutes. Zebrafish exposed to anatoxin-a contaminated water had altered heart rates.<ref>{{cite journal | vauthors = Ferrão-Filho A, Kozlowsky-Suzuki B | title = Cyanotoxins: bioaccumulation and effects on aquatic animals | journal = Marine Drugs | volume = 9 | issue = 12 | pages = 2729–72 | date = December 2011 | pmid = 22363248 | pmc = 3280578 | doi = 10.3390/md9122729 | doi-access = free }}</ref>

There have been cases of non-lethal poisoning in humans who have ingested water from streams and lakes that contain various genera of cyanobacteria that are capable of producing anatoxin-a. The effects of non-lethal poisoning were primarily gastrointestinal: nausea, vomiting, diarrhea, and abdominal pain.<ref>{{cite book | vauthors = Schwimmer D, Schwimmer M |chapter =Algae and Medicine|date=1964|url=http://link.springer.com/10.1007/978-1-4684-1719-7_17|title=Algae and Man|pages=368–412| veditors = Jackson DF |place=Boston, MA|publisher=Springer US|language=en|doi=10.1007/978-1-4684-1719-7_17|isbn=978-1-4684-1721-0|access-date=2020-10-25 }}</ref> A case of lethal poisoning was reported in Wisconsin after a teen jumped into a pond contaminated with cyanobacteria.<ref>{{Cite journal|last=Weirich CA, Miller TR|date=2014|title=Freshwater harmful algal blooms: toxins and children's health|url=|journal=Current Problems in Pediatric and Adolescent Health Care|volume=44|issue=1|pages=2–24|doi=10.1016/j.cppeds.2013.10.007|pmid=24439026}}</ref>

=== Exposure routes ===

==== Oral ====

Ingestion of drinking water or recreational water that is contaminated with anatoxin-a can pose fatal consequences since anatoxin-a was found to be quickly absorbed through the gastrointestinal tract in animal studies.<ref>{{cite journal| vauthors = Taylor JA |date=1995|title=A review of: "Detection Methods for Cyanobacterial Toxins" |journal=Chemistry and Ecology|language=en|volume=11|issue=4|pages=275–276|doi=10.1080/02757549508039077|bibcode=1995ChEco..11..275T |issn=0275-7540}}</ref> Dozens of cases of animal deaths due to ingestion of anatoxin-a contaminated water from lakes or rivers have been recorded, and it is suspected to have also been the cause of death of one human.<ref name="ofmpub.epa.gov">{{cite report | title = Toxicological Reviews of Cyanobacterial Toxins: Anatoxin-A | work = National Center for Environmental Assessment | publisher = U.S. Environmental Protection Agency | date = November 2006 |url=http://ofmpub.epa.gov/eims/eimscomm.getfile?p_download_id=459566 |access-date=2018-09-22 |archive-url=https://web.archive.org/web/20180923005549/https://ofmpub.epa.gov/eims/eimscomm.getfile?p_download_id=459566 |archive-date=2018-09-23 }}</ref> One study found that anatoxin-a is capable of binding to acetylcholine receptors and inducing toxic effects with concentrations in the nano-molar (nM) range if ingested.<ref>{{cite journal| vauthors = Wonnacott S, Gallagher T |date=2006-04-06|title=The Chemistry and Pharmacology of Anatoxin-a and Related Homotropanes with respect to Nicotinic Acetylcholine Receptors|journal=Marine Drugs|language=en|volume=4|issue=3|pages=228–254|doi=10.3390/md403228|s2cid=14060293|doi-access=free}}</ref>

==== Dermal ====

Dermal exposure is the most likely form of contact with cyanotoxins in the environment. Recreational exposure to river, stream, and lake waters contaminated with algal blooms has been known to cause skin irritation and rashes.<ref>{{cite journal | vauthors = Kaminski A, Bober B, Chrapusta E, Bialczyk J | title = Phytoremediation of anatoxin-a by aquatic macrophyte Lemna trisulca L | journal = Chemosphere | volume = 112 | pages = 305–10 | date = October 2014 | pmid = 25048920 | doi = 10.1016/j.chemosphere.2014.04.064 | bibcode = 2014Chmsp.112..305K }}</ref> The first study that looked at ''[[in vitro]]'' [[Cytotoxicity|cytotoxic]] effects of anatoxin-a on human [[Keratinocyte|skin cell]] proliferation and migration found that anatoxin-a exerted no effect at 0.1&nbsp;μg/mL or 1&nbsp;μg/mL, and a weak toxic effect at 10&nbsp;μg/mL only after an extended period of contact (48 hours).<ref>{{cite journal | vauthors = Adamski M, Zimolag E, Kaminski A, Drukała J, Bialczyk J | title = Effects of cylindrospermopsin, its decomposition products, and anatoxin-a on human keratinocytes | journal = The Science of the Total Environment | page = 142670 | date = October 2020 | volume = 765 | pmid = 33069473 | doi = 10.1016/j.scitotenv.2020.142670 | s2cid = 224779396 }}</ref>

==== Inhalation ====

No data on inhalation toxicity of anatoxin-a is currently available, though severe [[Shortness of breath|respiratory distress]] occurred in a water skier after they inhaled water spray containing a fellow cyanobacterial neurotoxin, [[saxitoxin]].<ref>{{cite journal| vauthors = Falconer IR |date=1996|title=Potential impact on human health of toxic cyanobacteria 1|journal=Phycologia|language=en|volume=35|issue=sup6|pages=6–11|doi=10.2216/i0031-8884-35-6S-6.1|bibcode=1996Phyco..35S...6F |issn=0031-8884}}</ref> It is possible that inhalation of water spray containing anatoxin-a could pose similar consequences.

===Mechanism of toxicity===

Anatoxin-a is an agonist of both neuronal α₄β₂ and α₄ [[nicotinic acetylcholine receptor]]s present in the CNS as well as the (α₁)₂βγδ muscle-type nAchRs that are present at the [[neuromuscular junction]].<ref name="Aráoz" /> (Anatoxin-a has an affinity for these muscle-type receptors that is about 20 times greater than that of [[acetylcholine]].<ref name="Botana" />) However, the cyanotoxin has little effect on [[muscarinic acetylcholine receptors]]; it has a 100 fold lesser selectivity for these types of receptors than it has for nAchRs.<ref name="Osswald">{{cite journal|vauthors=Osswald J, Rellán S, Gago A, Vasconcelos V|date=November 2007|title=Toxicology and detection methods of the alkaloid neurotoxin produced by cyanobacteria, anatoxin-a|journal=Environment International|volume=33|issue=8|pages=1070–89|doi=10.1016/j.envint.2007.06.003|pmid=17673293|bibcode=2007EnInt..33.1070O }}</ref> Anatoxin-a also shows much less potency in the CNS than in neuromuscular junctions. In hippocampal and brain stem neurons, a 5 to 10 times greater concentration of anatoxin-a was necessary to activate nAchRs than what was required in the PNS.<ref name="Osswald" />

In normal circumstances, [[acetylcholine]] binds to nAchRs in the post-synaptic neuronal membrane, causing a conformational change in the extracellular domain of the receptor which in turn opens the channel pore. This allows Na⁺ and Ca²⁺ ions to move into the neuron, causing cell [[depolarization]] and inducing the generation of [[action potential]]s, which allows for muscle contraction. The acetylcholine neurotransmitter then dissociates from the nAchR, where it is rapidly cleaved into [[acetate]] and [[choline]] by [[acetylcholinesterase]].<ref>{{cite book | vauthors = Purves D, Augustine G, Fitzpatrick D, Hall W, Lamantia AS, White L | title = Neuroscience | edition = 5th | location = Sunderland, Massachusetts | publisher = Sinauer Associates, Inc. | date = 2012 }}</ref>

[[File:Anatoxina.png|thumb|The effects of anatoxin-a on nicotinic acetylcholine receptors at the neuromuscular junction]]

Anatoxin-a binding to these nAchRs cause the same effects in neurons. However, anatoxin-a [[Irreversible agonist|binding is irreversible]], and the anatoxin-a nAchR complex cannot be broken down by [[acetylcholinesterase]]. Thus, the nAchR is temporarily locked open, which leads to overstimulation due to the constant generation of action potentials.<ref name="Osswald" />

Two enantiomers of anatoxin-a, the positive [[enantiomer]], (+)-anatoxin-a, is 150 fold more potent than the synthetic negative enantiomer, (−)-anatoxin-a.<ref name="Osswald" /> This is because (+)-anatoxin-a, the s-''cis'' enone conformation, has a distance a 6.0 [[Angstrom (unit)|Å]] between its [[nitrogen]] and [[carbonyl]] group, which corresponds well to the 5.9 Å distance that separate the nitrogen and oxygen in acetylcholine.<ref name="Aráoz" />

[[Respiratory arrest]], which results in a lack of an oxygen supply to the brain, is the most evident and lethal effect of anatoxin-a.<ref name="Osswald" /> Injections of mice, rats, birds, dogs, and calves with lethal doses of anatoxin-a have demonstrated that death is preceded by a sequence of muscle [[fasciculations]], decreased movement, collapse, exaggerated abdominal breathing, [[cyanosis]] and [[convulsions]].<ref name="Botana" /> In mice, anatoxin-a also seriously impacted blood pressure and heart rate, and caused severe [[acidosis]].<ref name="Aráoz" />

=== Cases of toxicity ===

[[File:Flickr - Rainbirder - Born of Fire.jpg|thumb|left|Flamingos at Lake Bogoria]]

Many cases of wildlife and livestock deaths due to anatoxin-a have been reported since its discovery. Domestic dog deaths due to the cyanotoxin, as determined by analysis of stomach contents, have been observed at the lower North Island in New Zealand in 2005,<ref>{{cite journal | vauthors = Wood SA, Selwood AI, Rueckert A, Holland PT, Milne JR, Smith KF, Smits B, Watts LF, Cary CS | title = First report of homoanatoxin-a and associated dog neurotoxicosis in New Zealand | journal = Toxicon | volume = 50 | issue = 2 | pages = 292–301 | date = August 2007 | pmid = 17517427 | doi = 10.1016/j.toxicon.2007.03.025 }}</ref> in eastern France in 2003,<ref>{{cite journal | vauthors = Gugger M, Lenoir S, Berger C, Ledreux A, Druart JC, Humbert JF, Guette C, Bernard C | title = First report in a river in France of the benthic cyanobacterium Phormidium favosum producing anatoxin-a associated with dog neurotoxicosis | journal = Toxicon | volume = 45 | issue = 7 | pages = 919–28 | date = June 2005 | pmid = 15904687 | doi = 10.1016/j.toxicon.2005.02.031 }}</ref> in California of the United States in 2002 and 2006,<ref>{{cite journal | vauthors = Puschner B, Hoff B, Tor ER | title = Diagnosis of anatoxin-a poisoning in dogs from North America | journal = Journal of Veterinary Diagnostic Investigation | volume = 20 | issue = 1 | pages = 89–92 | date = January 2008 | pmid = 18182518 | doi = 10.1177/104063870802000119 | doi-access = free }}</ref> in Scotland in 1992, in Ireland in 1997 and 2005,<ref name="Botana" /> in Germany in 2017<ref>{{Cite journal|last1=Fastner|first1=Jutta|last2=Beulker|first2=Camilla|last3=Geiser|first3=Britta|last4=Hoffmann|first4=Anja|last5=Kröger|first5=Roswitha|last6=Teske|first6=Kinga|last7=Hoppe|first7=Judith|last8=Mundhenk|first8=Lars|last9=Neurath|first9=Hartmud|last10=Sagebiel|first10=Daniel|last11=Chorus|first11=Ingrid|date=February 2018|title=Fatal Neurotoxicosis in Dogs Associated with Tychoplanktic, Anatoxin-a Producing Tychonema sp. in Mesotrophic Lake Tegel, Berlin|journal=Toxins|language=en|volume=10|issue=2|page=60|doi=10.3390/toxins10020060|pmid=29385106|pmc=5848161|doi-access=free}}</ref> and 2020.<ref>{{Cite journal|pmc=7699839|year = 2020|last1 = Bauer|first1 = F.|last2 = Fastner|first2 = J.|last3 = Bartha-Dima|first3 = B.|last4 = Breuer|first4 = W.|last5 = Falkenau|first5 = A.|last6 = Mayer|first6 = C.|last7 = Raeder|first7 = U.|title = Mass Occurrence of Anatoxin-a- and Dihydroanatoxin-a-Producing Tychonema sp. In Mesotrophic Reservoir Mandichosee (River Lech, Germany) as a Cause of Neurotoxicosis in Dogs|journal = Toxins|volume = 12|issue = 11|page = 726|doi = 10.3390/toxins12110726|pmid = 33233760|doi-access = free}}</ref> In each case, the dogs began showing muscle convulsions within minutes, and were dead within a matter of hours. Numerous cattle fatalities arising from the consumption of water contaminated with cyanobacteria that produce anatoxin-a have been reported in the United States, Canada, and Finland between 1980 and the present.<ref name="Botana" />

A particularly interesting case of anatoxin-a poisoning is that of [[lesser flamingos]] at [[Lake Bogoria]] in [[Kenya]]. The cyanotoxin, which was identified in the stomachs and fecal pellets of the birds, killed roughly 30,000 flamingos in the second half of 1999, and continues to cause mass fatalities annually, devastating the flamingo population. The toxin is introduced into the birds via water contaminated with cyanobacterial mat communities that arise from the hot springs in the lake bed.<ref>{{cite journal | vauthors = Krienitz L, Ballot A, Kotut K, Wiegand C, Pütz S, Metcalf JS, Codd GA, Pflugmacher S | title = Contribution of hot spring cyanobacteria to the mysterious deaths of Lesser Flamingos at Lake Bogoria, Kenya | journal = FEMS Microbiology Ecology | volume = 43 | issue = 2 | pages = 141–8 | date = March 2003 | pmid = 19719674 | doi = 10.1111/j.1574-6941.2003.tb01053.x | doi-access = free | bibcode = 2003FEMME..43..141K }}</ref>

==Synthesis==

=== Laboratory synthesis ===

====Cyclic expansion of tropanes====

The first biologically occurring initial substance for [[tropane]] expansion into anatoxin-a was [[cocaine]], which has similar stereochemistry to anatoxin-a. Cocaine is first converted into the endo isomer of a cyclopropane, which is then photolytically cleaved to obtain an alpha, beta unsaturated ketone. Through the use of diethyl azodicarboxylate, the ketone is demethylated and anatoxin-a is formed. A similar, more recent synthesis pathway involves producing 2-tropinone from cocaine and treating the product with ethyl chloroformate producing a bicyclic ketone. This product is combined with trimethylsilyldiazylmethane, an organoaluminum Lewis acid and trimethylsinyl enol ether to produce tropinone. This method undergoes several more steps, producing useful intermediates as well as anatoxin-a as a final product.<ref name="Botana" /> [[File:Kokain - Cocaine.svg|thumb|alt=Cocaine, a precursor for anatoxin-a synthesis.|Cocaine, a precursor for anatoxin-a synthesis]]

====Cyclization of cyclooctenes====

The first and most extensively explored approach used to synthesize anatoxin-a in vitro, cyclooctene cyclization involves 1,5-cyclooctadiene as its initial source. This starting substance is reacted to form methyl amine and combined with hypobromous acid to form anatoxin-a. Another method developed in the same laboratory uses aminoalcohol in conjunction with mercuric (II) acetate and sodium borohydride. The product of this reaction was transformed into an alpha, beta ketone and oxidized by ethyl azodicarboxylate to form anatoxin-a.<ref name="Botana" />

====Enantioselective enolization strategy====

This method for anatoxin-a production was one of the first used that does not utilize a chimerically analogous starting substance for anatoxin formation. Instead, a racemic mixture of 3-tropinone is used with a chiral lithium amide base and additional ring expansion reactions in order to produce a ketone intermediate. Addition of an organocuprate to the ketone produces an enol triflate derivative, which is then lysed hydrogenously and treated with a deprotecting agent in order to produce anatoxin-a. Similar strategies have also been developed and utilized by other laboratories.<ref name="Botana" />

====Intramolecular cyclization of iminium ions====

Iminium ion cyclization utilizes several different pathways to create anatoxin-a, but each of these produces and progresses with a pyrrolidine iminium ion. The major differences in each pathway relate to the precursors used to produce the imium ion and the total yield of anatoxin-a at the end of the process. These separate pathways include production of alkyl iminium salts, acyl iminium salts and tosyl iminium salts.<ref name="Botana" />

====Enyne metathesis====

Enyne metathesis of anatoxin-a involves the use of a ring closing mechanism and is one of the more recent advances in anatoxin-a synthesis. In all methods involving this pathway, pyroglutamic acid is used as a starting material in conjunction with a Grubb's catalyst. Similar to iminium cyclization, the first attempted synthesis of anatoxin-a using this pathway used a 2,5-cis-pyrrolidine as an intermediate.<ref name="Botana" />

== Biosynthesis ==

Anatoxin-a is synthesized ''in vivo'' in the species ''Anabaena flos-aquae'',<ref name="Botana" /> as well as several other genera of cyanobacteria. Anatoxin-a and related chemical structures are produced using acetate and glutamate. Further enzymatic reduction of these precursors results in the formation of anatoxin-a. Homoanatoxin, a similar chemical, is produced by ''Oscillatoria formosa'' and utilizes the same precursor. However, homoanatoxin undergoes a methyl addition by S-adenosyl-L-methionine instead of an addition of electrons, resulting in a similar analogue.<ref name="Aráoz" /> The biosynthetic gene cluster (BGC) for anatoxin-a was described from ''[[Oscillatoria]]'' PCC 6506 in 2009.<ref name="Méjean2009">{{cite journal | last1=Méjean | first1=Annick | last2=Mann | first2=Stéphane | last3=Maldiney | first3=Thomas | last4=Vassiliadis | first4=Gaëlle | last5=Lequin | first5=Olivier | last6=Ploux | first6=Olivier | title=Evidence that Biosynthesis of the Neurotoxic Alkaloids Anatoxin-a and Homoanatoxin-a in the Cyanobacterium Oscillatoria PCC 6506 Occurs on a Modular Polyketide Synthase Initiated by l-Proline | journal=Journal of the American Chemical Society | publisher=American Chemical Society (ACS) | volume=131 | issue=22 | date=2009-05-13 | issn=0002-7863 | doi=10.1021/ja9024353 | pages=7512–7513| pmid=19489636 }}</ref>

==Stability and degradation==

Anatoxin-a is unstable in water and other natural conditions, and in the presence of UV light undergoes [[photodegradation]], being converted to the less toxic products dihydroanatoxin-a and epoxyanatoxin-a. The photodegradation of anatoxin-a is dependent on pH and sunlight intensity but independent of oxygen, indicating that the degradation by light is not achieved through the process of photo-oxidation.<ref name="Osswald" />

Studies have shown that some microorganisms are capable of degrading anatoxin-a. A study done by Kiviranta and colleagues in 1991 showed that the bacterial genus ''[[Pseudomonas]]'' was capable of degrading anatoxin-a at a rate of 2–10 μg/ml per day.<ref>{{cite journal | vauthors = Kiviranta J, Sivonen K, Lahti K, Luukkainen R, Niemelä SI | title = Production and biodegradation of cyanobacterial toxins-a laboratory study. | journal = Archiv für Hydrobiologie | date = 1991 | volume = 121 | issue = 3 | pages = 281–94 | doi = 10.1127/archiv-hydrobiol/121/1991/281 | s2cid = 88901836 |url=https://researchportal.helsinki.fi/en/publications/production-and-biodegradation-of-cyanobacterial-toxins-a-laborato }}</ref> Later experiments done by Rapala and colleagues (1994) supported these results. They compared the effects of sterilized and non-sterilized sediments on anatoxin-a degradation over the course of 22 days, and found that after that time vials with the sterilized sediments showed similar levels of anatoxin-a as at the commencement of the experiment, while vials with non-sterilized sediment showed a 25-48% decrease.<ref name="Osswald" />

==Detection==

There are two categories of anatoxin-a detection methods. Biological methods have involved administration of samples to mice and other organisms more commonly used in ecotoxicological testing, such as [[brine shrimp]] (''Artemia salina''), larvae of the freshwater crustacean ''[[Thamnocephalus platyurus]]'', and various insect larvae. Problems with this methodology include an inability to determine whether it is anatoxin-a or another neurotoxin that causes the resulting deaths. Large amounts of sample material are also needed for such testing. In addition to the biological methods, scientists have used [[chromatography]] to detect anatoxin-a. This is complicated by the rapid degradation of the toxin and the lack of commercially available standards for anatoxin-a.<ref name="Osswald" />

==Public health==

Despite the relatively low frequency of anatoxin-a relative to other cyanotoxins, its high toxicity (the lethal dose is not known for humans, but is estimated to be less than 5&nbsp;mg for an adult male<ref>{{cite journal | vauthors = Patockaa J, Stredab L | title = Brief review of natural nonprotein neurotoxins. | journal = ASA Newsletter | date = 2002 | volume = 89 | issue = 2 | pages = 16–24 |url=http://www.asanltr.com/newsletter/02-2/articles/Neurotoxins.htm |archive-url=https://web.archive.org/web/20130104141238/http://www.asanltr.com/newsletter/02-2/articles/Neurotoxins.htm |archive-date=2013-01-04 }}</ref>) means that it is still considered a serious threat to terrestrial and aquatic organisms, most significantly to livestock and to humans. Anatoxin-a is suspected to have been involved in the death of at least one person.<ref name="ofmpub.epa.gov"/> The threat posed by anatoxin-a and other cyanotoxins is increasing as both fertilizer runoff, leading to [[eutrophication]] in lakes and rivers, and higher global temperatures contribute to a greater frequency and prevalence of cyanobacterial blooms.<ref name="Osswald" />

=== Water regulations ===

The [[World Health Organization]] in 1999 and [[United States Environmental Protection Agency|EPA]] in 2006 both came to the conclusion that there was not enough toxicity data for anatoxin-a to establish a formal tolerable daily intake (TDI) level, though some places have implemented levels of their own.<ref name=":1">{{cite web | date=June 2015|title=2015 Drinking Water Health Advisories for Two Cyanobacterial Toxins|url=https://19january2017snapshot.epa.gov/sites/production/files/2015-06/documents/cyanotoxins-fact_sheet-2015.pdf|access-date=October 25, 2020|website=United States Environmental Protection Agency}}</ref><ref>{{cite book|title=Toxic cyanobacteria in water: a guide to their public health consequences, monitoring, and management|date=1999|publisher=E & FN Spon|editor=Chorus, Ingrid |editor2=Bartram, Jamie |isbn=0-419-23930-8|location=London|oclc=40395794}}</ref>

==== United States ====

===== Drinking water advisory levels =====

Anatoxin-a is not regulated under the [[Safe Drinking Water Act]], but states are allowed to create their own standards for contaminants that are unregulated. Currently there are four states that have set drinking water advisory levels for anatoxin-a as seen in the table below.<ref>{{cite web|date=2018-02-12|title=Rules and Regulations: Drinking Water HABs Response Plan|url=https://deq.utah.gov/drinking-water/rules-and-regulations-habs|access-date=2020-10-14|website=Utah Department of Environmental Quality|language=en-US}}</ref> On October 8, 2009 the EPA published the third Drinking Water [[Contaminant candidate list|Contaminant Candidate List]] (CCL) which included anatoxin-a (among other cyanotoxins), indicating that anatoxin-a may be present in public water systems but is not regulated by the EPA. Anatoxin-a's presence on the CCL means that it may need to be regulated by the EPA in the future, pending further information on its health effects in humans.<ref>{{cite web|date=2009-10-08|title=Drinking Water Contaminant Candidate List 3-Final|url=https://www.federalregister.gov/documents/2009/10/08/E9-24287/drinking-water-contaminant-candidate-list-3-final|access-date=2020-09-27|website=Federal Register}}</ref><ref name=":1" />

{| class="wikitable"

|+Drinking Water Advisory Levels

!State

!Concentration (μg/L)

|-

|Minnesota

|0.1

|-

|Ohio

|20

|-

|Oregon

|0.7

|-

|Vermont

|0.5

|}

===== Recreational water advisory levels =====

In 2008 the state of Washington implemented a recreational advisory level for anatoxin-a of 1&nbsp;μg/L in order to better manage algal blooms in lakes and protect users from exposure to the blooms.<ref>{{cite web | date=July 2008|title=Washington State Recreational Guidance for Microcystins (Provisional) and Anatoxin-a (Interim/Provisional)|url=https://www.doh.wa.gov/portals/1/documents/4400/334-177-recguide.pdf|access-date=October 25, 2020|website=Washington State Department of Health}}</ref>

==== Canada ====

The Canadian province of Québec has a drinking water Maximum Accepted Value for anatoxin-a of 3.7&nbsp;μg/L.<ref>{{cite journal | vauthors = Carrière A, Prévost M, Zamyadi A, Chevalier P, Barbeau B | title = Vulnerability of Quebec drinking-water treatment plants to cyanotoxins in a climate change context | journal = Journal of Water and Health | volume = 8 | issue = 3 | pages = 455–65 | date = September 2010 | pmid = 20375475 | doi = 10.2166/wh.2009.207 | url = https://iwaponline.com/jwh/article/8/3/455/18125/Vulnerability-of-Quebec-drinkingwater-treatment | doi-access = free }}</ref>

==== New Zealand ====

New Zealand has a drinking water Maximum Accepted Value for anatoxin-a of 6&nbsp;μg/L.<ref>{{cite journal | vauthors = Merel S, Walker D, Chicana R, Snyder S, Baurès E, Thomas O | title = State of knowledge and concerns on cyanobacterial blooms and cyanotoxins | journal = Environment International | volume = 59 | pages = 303–27 | date = September 2013 | pmid = 23892224 | doi = 10.1016/j.envint.2013.06.013 | doi-access = free | bibcode = 2013EnInt..59..303M }}</ref>

===Water treatment===

As of now, there is no official guideline level for anatoxin-a,<ref name="Ho">{{cite journal | vauthors = Ho L, Sawade E, Newcombe G | title = Biological treatment options for cyanobacteria metabolite removal--a review | journal = Water Research | volume = 46 | issue = 5 | pages = 1536–48 | date = April 2012 | pmid = 22133838 | doi = 10.1016/j.watres.2011.11.018 }}</ref> although scientists estimate that a level of 1 μg l⁻¹ would be sufficiently low.<ref name="pmid10215107">{{cite journal | vauthors = Fawell JK, Mitchell RE, Hill RE, Everett DJ | title = The toxicity of cyanobacterial toxins in the mouse: II anatoxin-a | journal = Human & Experimental Toxicology | volume = 18 | issue = 3 | pages = 168–73 | date = March 1999 | pmid = 10215107 | doi = 10.1177/096032719901800306 | bibcode = 1999HETox..18..168F | s2cid = 38639505 }}</ref> Likewise, there are no official guidelines regarding testing for anatoxin-a. Among methods of reducing the risk for cyanotoxins, including anatoxin-a, scientists look favorably on biological treatment methods because they do not require complicated technology, are low maintenance, and have low running costs. Few biological treatment options have been tested for anatoxin-a specifically, although a species of ''Pseudomonas'', capable of biodegrading anatoxin-a at a rate of 2–10 μg ml⁻¹ d⁻¹, has been identified. Biological (granular) [[activated carbon]] (BAC) has also been tested as a method of biodegradation, but it is inconclusive whether biodegradation occurred or if anatoxin-a was simply adsorbing the activated carbon.<ref name="Ho" /> Others have called for additional studies to determine more about how to use activated carbon effectively.<ref name="Westrick">{{cite journal | vauthors = Westrick JA, Szlag DC, Southwell BJ, Sinclair J | title = A review of cyanobacteria and cyanotoxins removal/inactivation in drinking water treatment | journal = Analytical and Bioanalytical Chemistry | volume = 397 | issue = 5 | pages = 1705–14 | date = July 2010 | pmid = 20502884 | doi = 10.1007/s00216-010-3709-5 | s2cid = 206903692 }}</ref>

Chemical treatment methods are more common in drinking water treatment compared to biological treatment, and numerous processes have been suggested for anatoxin-a. [[oxidizing agent|Oxidants]] such as [[potassium permanganate]], [[ozone]], and advanced oxidation processes ([[Advanced oxidation process|AOPs]]) have worked in lowering levels of anatoxin-a, but others, including photocatalysis, UV [[photolysis]],<ref name="Westrick" /> and [[Water chlorination|chlorination]],<ref>{{cite journal | vauthors = Merel S, Clément M, Thomas O | title = State of the art on cyanotoxins in water and their behaviour towards chlorine | journal = Toxicon | volume = 55 | issue = 4 | pages = 677–91 | date = April 2010 | pmid = 19874838 | doi = 10.1016/j.toxicon.2009.10.028 }}</ref> have not shown great efficacy.

Directly removing the cyanobacteria in the water treatment process through physical treatment (e.g., [[Membrane technology|membrane filtration]]) is another option because most of the anatoxin-a is contained within the cells when the bloom is growing. However, anatoxin-a is released from cyanobacteria into water when they [[Senescence|senesce]] and lyse, so physical treatment may not remove all of the anatoxin-a present.<ref>{{cite journal | vauthors = Bouma-Gregson K, Kudela RM, Power ME | title = Widespread anatoxin-a detection in benthic cyanobacterial mats throughout a river network | journal = PLOS ONE | volume = 13 | issue = 5 | pages = e0197669 | date = 2018-05-18 | pmid = 29775481 | pmc = 5959195 | doi = 10.1371/journal.pone.0197669 | bibcode = 2018PLoSO..1397669B | veditors = Humbert JF | doi-access = free }}</ref> Additional research needs to be done to find more reliable and efficient methods of both detection and treatment.<ref name="Westrick" />

==Laboratory uses==

Anatoxin-a is a very powerful nicotinic acetylcholine receptor agonist and as such has been extensively studied for medicinal purposes. It is mainly used as a pharmacological probe in order to investigate diseases characterized by low acetylcholine levels, such as [[muscular dystrophy]], [[myasthenia gravis]], [[Alzheimer disease]], and [[Parkinson disease]]. Further research on anatoxin-a and other less potent analogues are being tested as possible replacements for acetylcholine.<ref name="Botana" />

==Genera of cyanobacteria that produce anatoxin-a==

*''[[Anabaena]] (Dolichospermum)''<ref>{{cite web|last=Australian Water Quality Centre|date=2015-12-04|title=Notification of Recent Name Changes for Cyanobacteria Adopted and Reported by AWQC|url=https://www.awqc.com.au/news/notification-of-recent-name-changes-for-cyanobacteria-adopted-and-reported-by-awqc|access-date=2020-10-15|website=www.awqc.com.au|language=en-AU}}</ref>

*''[[Aphanizomenon]]''<ref name=":1" />

*''[[Cylindrospermopsis]]''<ref name=":0">{{cite journal | vauthors = Christensen VG, Khan E | title = Freshwater neurotoxins and concerns for human, animal, and ecosystem health: A review of anatoxin-a and saxitoxin | journal = The Science of the Total Environment | volume = 736 | page = 139515 | date = September 2020 | pmid = 32485372 | doi = 10.1016/j.scitotenv.2020.139515 | bibcode = 2020ScTEn.736m9515C | s2cid = 219288601 }}</ref>

*''[[Cylindrospermum]]''

*''[[Lyngbya]]''<ref name="Paerl_2013">{{cite journal | vauthors = Paerl HW, Otten TG | title = Harmful cyanobacterial blooms: causes, consequences, and controls | journal = Microbial Ecology | volume = 65 | issue = 4 | pages = 995–1010 | date = May 2013 | pmid = 23314096 | doi = 10.1007/s00248-012-0159-y | bibcode = 2013MicEc..65..995P | s2cid = 5718333 | url = http://link.springer.com/10.1007/s00248-012-0159-y }}</ref>

*''[[Microcystis]]''<ref>{{cite journal | vauthors = Park HD, Watanabe MF, Harda K, Nagai H, Suzuki M, Watanabe M, Hayashi H | title = Hepatotoxin (microcystin) and neurotoxin (anatoxin-a) contained in natural blooms and strains of cyanobacteria from Japanese freshwaters | journal = Natural Toxins | volume = 1 | issue = 6 | pages = 353–60 | date = 1993 | pmid = 8167957 | doi = 10.1002/nt.2620010606 }}</ref>

*''[[Nostoc]]''<ref name=":0" />

*''[[Oscillatoria]]''<ref name="Paerl_2013" />

*''Microcoleus (Phormidium)''<ref name="Paerl_2013" />

*''[[Planktothrix]]''<ref name="Paerl_2013" />

*''Raphidiopsis''<ref name="Paerl_2013" />

*''Tychonema''<ref>{{cite journal | vauthors = Shams S, Capelli C, Cerasino L, Ballot A, Dietrich DR, Sivonen K, Salmaso N | title = Anatoxin-a producing Tychonema (Cyanobacteria) in European waterbodies | journal = Water Research | volume = 69 | pages = 68–79 | date = February 2015 | pmid = 25437339 | doi = 10.1016/j.watres.2014.11.006 | bibcode = 2015WatRe..69...68S | url = http://nbn-resolving.de/urn:nbn:de:bsz:352-0-286858 }}</ref>

*''Woronichinia''<ref name="Paerl_2013" />

== See also ==

* [[Guanitoxin]]

* [[Epibatidine]]

{{reflist}}

== Further reading ==

{{refbegin}}

* {{cite journal | vauthors = Wood SA, Rasmussen JP, Holland PT, Campbell R, Crowe AL | year = 2007 | title = First Report of the Cyanotoxin Anatoxin-A from Aphanizomenon issatschenkoi (cyanobacteria) | journal = Journal of Phycology | volume = 43 | issue = 2| pages = 356–365 | doi=10.1111/j.1529-8817.2007.00318.x| s2cid = 84284928 }}

* {{cite journal | vauthors = Wonnacott S, Gallagher T | title = The Chemistry and Pharmacology of Anatoxin-a and Related Homotropanes with respect to Nicotinic Acetylcholine Receptors. | journal = Marine Drugs | date = April 2006 | volume = 4 | issue = 3 | pages = 228–254 | doi = 10.3390/md403228 | pmc = 3663412 | doi-access = free }}

{{refend}}

== External links ==

* [http://www.periodicvideos.com/videos/mv_veryfastdeathfactor.htm Very Fast Death Factor (Anatoxin-a)] at ''[[The Periodic Table of Videos]]'' (University of Nottingham)

* [http://www.chm.bris.ac.uk/motm/antx/antx.htm Molecule of the Month: Anatoxin] at the School of Chemistry, Physics, and Environmental Studies, University of Sussex at Brighton

{{Cyanotoxins}}

{{Neurotoxins}}

{{Nicotinic acetylcholine receptor modulators}}

[[Category:Cyanotoxins]]

[[Category:Cycloalkenes]]

[[Category:Amines]]

[[Category:Heterocyclic compounds with 2 rings]]

[[Category:Enones]]

[[Category:Bacterial alkaloids]]

[[Category:Nicotinic agonists]]