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 loss of coordination, muscular fasciculations, convulsions 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.
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.
Occurrence Anatoxin-a is a neurotoxin produced by multiple genera of freshwater cyanobacteria that are found in water bodies globally. 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. Blooms of cyanobacteria that produce anatoxin-a among other cyanotoxins are increasing in frequency due to increasing temperatures, stratification, and eutrophication due to nutrient runoff. These expansive cyanobacterial harmful algal blooms, known as cyanoHABs, increase the amount of cyanotoxins in the surrounding water, threatening the health of both aquatic and terrestrial organisms. Some species of cyanobacteria that produce anatoxin-a don't produce surface water blooms but instead form benthic mats. Many cases of anatoxin-a related animal deaths have occurred due to ingestion of detached benthic cyanobacterial mats that have washed ashore.
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.
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 mg/kg body weight.
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. They determined the LD50 for mice to be 250 μg/kg body weight.
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.
Toxicity
Effects Laboratory studies using mice showed that characteristic effects of acute anatoxin-a poisoning via intraperitoneal injection include muscle fasciculations, tremors, staggering, gasping, respiratory paralysis, and death within minutes. Zebrafish exposed to anatoxin-a contaminated water had altered heart rates.
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. A case of lethal poisoning was reported in Wisconsin after a teen jumped into a pond contaminated with cyanobacteria.
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. 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. 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.
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. The first study that looked at in vitro cytotoxic effects of anatoxin-a on human skin cell proliferation and migration found that anatoxin-a exerted no effect at 0.1 μg/mL or 1 μg/mL, and a weak toxic effect at 10 μg/mL only after an extended period of contact (48 hours).
Inhalation No data on inhalation toxicity of anatoxin-a is currently available, though severe respiratory distress occurred in a water skier after they inhaled water spray containing a fellow cyanobacterial neurotoxin, saxitoxin. 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 receptors present in the CNS as well as the (α1)2βγδ muscle-type nAchRs that are present at the neuromuscular junction. (Anatoxin-a has an affinity for these muscle-type receptors that is about 20 times greater than that of acetylcholine.) 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. 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.
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 potentials, which allows for muscle contraction. The acetylcholine neurotransmitter then dissociates from the nAchR, where it is rapidly cleaved into acetate and choline by acetylcholinesterase.
Anatoxin-a binding to these nAchRs cause the same effects in neurons. However, anatoxin-a 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.
Two enantiomers of anatoxin-a, the positive enantiomer, (+)-anatoxin-a, is 150 fold more potent than the synthetic negative enantiomer, (−)-anatoxin-a.
Respiratory arrest, which results in a lack of an oxygen supply to the brain, is the most evident and lethal effect of anatoxin-a. 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. In mice, anatoxin-a also seriously impacted blood pressure and heart rate, and caused severe acidosis.
Cases of toxicity
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, in eastern France in 2003, in California of the United States in 2002 and 2006, in Scotland in 1992, in Ireland in 1997 and 2005, in Germany in 2017 and 2020. 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.
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.
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.
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.
Enantioselective enolization strategy