Cylindrospermopsin
Encyclopedia
Cylindrospermopsin is a cyanotoxin
produced by a variety of freshwater
cyanobacteria. CYN is a polycyclic
uracil
derivative
containing guanidino
and sulfate
groups. It is also zwitterion
ic, making it highly water soluble. CYN is toxic
to liver
and kidney
tissue and is thought to inhibit protein synthesis
and to covalently modify DNA
and/or RNA
. It is not known whether cylindrospermopsin is a carcinogen, but it appears to have no tumour initiating activity in mice.
CYN was first discovered after an outbreak of a mystery disease on Palm Island
, Queensland
, Australia
. The outbreak was traced back to a bloom
of Cylindrospermopsis raciborskii in the local drinking water supply, and the toxin was subsequently identified. Analysis of the toxin led to a proposed chemical structure
in 1992, which was revised after synthesis
was achieved in 2000. Several analogues of CYN, both toxic and non-toxic, have been isolated or synthesised.
C. raciborskii has been observed mainly in tropical areas, however has also recently been discovered in temperate regions of Australia
, Europe
, North
and South America
, and New Zealand
.
, Queensland
, Australia
, were admitted to hospital, suffering various symptoms of gastroenteritis
. All of these were children; in addition, 10 adults were affected but not hospitalised. Initial symptoms, including abdominal
pain and vomiting
, resembled those of hepatitis
; later symptoms included kidney failure and bloody diarrhoea. Urine
analysis revealed high levels of proteins, ketones and sugar
in many patients, along with blood
and urobilinogen
in lesser numbers. The urine analysis, along with faecal microscopy
and poison
screening, could not provide a statistical link to the symptoms. All patients recovered within 4 to 26 days, and at the time there was no apparent cause for the outbreak. Initial thoughts on the cause included poor water quality and diet, however none were conclusive, and the illness was coined the “Palm Island Mystery Disease”.
At the time, it was noticed that this outbreak coincided with a severe algal bloom in the local drinking water supply, and soon after the focus turned to the dam in question. An epidemiological study of this “mystery disease” later confirmed that the Solomon Dam was implicated, as those that became ill had used water from the dam. It became apparent that a recent treatment of the algal bloom
with copper sulfate caused lysis of the algal cells, releasing a toxin
into the water.
A study of the dam revealed that periodic blooms of algae were caused predominantly by three strains of cyanobacteria: two of the genus Anabaena
, and Cylindrospermopsis raciborskii, previously unknown in Australian waters. A mouse bioassay of the three demonstrated that although the two Anabaena strains were non-toxic, C. raciborskii was highly toxic. Later isolation of the compound responsible led to the identification of the toxin cylindrospermopsin.
A later report alternatively proposed that the excess copper in the water was the cause of the disease. The excessive dosing was following the use of least-cost contractors to control the algae, who were unqualified in the field.
Isolation of the toxin using algae cultured from the original Palm Island strain was achieved by gel filtration of an aqueous extract, followed by reverse-phase HPLC
. Structure elucidation was achieved via mass spectrometry
(MS) and nuclear magnetic resonance
(NMR) experiments, and a structure (later proven slightly incorrect) was proposed (Figure 1).
This almost-correct molecule
possesses a tricyclic
guanidine
group (rings A, B & C), along with a uracil
ring (D). The zwitterion
ic nature of the molecule makes this highly water-soluble, as the presence of charged
areas within the molecule creates a dipole
effect, suiting the polar
solvent
. Sensitivity of key signals in the NMR spectrum
to small changes in pH
suggested that the uracil ring exists in a keto
/enol
tautomer
ic relationship, where a hydrogen
transfer results in two distinct structures (Figure 2). It was originally proposed that a hydrogen bond
between the uracil and guanidine groups in the enol tautomer would make this the dominant form.
A second metabolite
of C. raciborskii was identified from extracts of the algae after the observation of a frequently occurring peak accompanying that of CYN during UV
and MS experiments. Analysis by MS and NMR methods concluded that this new compound was missing the oxygen
adjacent to the uracil ring, and was named deoxycylindrospermopsin (Figure 3).
In 1999, an epimer
of CYN, named 7-epicyclindrospermopsin (epiCYN), was also identified as a minor metabolite from Aphanizomenon ovalisporum. This occurred whilst isolating CYN from cyanobacteria taken from Lake Kinneret in Israel
. The proposed structure of this molecule differed from CYN only in the orientation of the hydroxyl
group adjacent to the uracil ring (Figure 4).
approaches to CYN started with the piperidine
ring (A), and progressed to annulation
of rings B and C. The first total synthesis
of CYN was reported in 2000 through a 20-step process.
Improvements to synthetic methods led to a revision of the stereochemistry
of CYN in 2001. A synthetic process controlling each of the six stereogenic centres of epiCYN established that the original assignments of both CYN and epiCYN were in fact a reversal of the correct structures. An alternative approach by White and Hansen supported these absolute configurations (Figure 5). At the time of this correct assignment, it was suggested that the enol form was not dominant.
of CYN is its stability
. Although the toxin has been found to degrade
rapidly in an algal extract when exposed to sunlight
, it is resistant to degradation by changes in pH
and temperature
, and shows no degradation in either the pure solid form or in pure water. As a result, in turbid
and unmoving water the toxin can persist for long periods, and although boiling water will kill the algae, it may not remove the toxin.
poisoned mice displayed anorexia
, diarrhoea and gasping respiration
. Autopsy results revealed haemorrhages in the lungs, livers, kidneys, small intestines and adrenal glands. Histopathology
revealed dose-related necrosis
of hepatocytes, lipid
accumulation, and fibrin
thrombi formation in blood vessels of the liver and lungs, along with varying epithelial cell necrosis in areas of the kidneys.
A more recent mouse bioassay of the effects of cylindrospermopsin revealed an increase in liver weight, with both lethal and non-lethal doses; in addition the livers appeared dark-coloured. Extensive necrosis of hepatocytes was visible in mice administered a lethal dose, and some localised damage was also observed in mice administered a non-lethal dose.
. A further experiment in 1997 measured the LD50 as 52 mg/kg at 24 hours and 32 mg/kg at 7 days, however the data suggested that another toxic compound was present in the isolate of sonicated
cells used; predictions made by Ohtani et al. about the 24 hour toxicity were considerably higher, and it was proposed that another metabolite was present to account for the relatively low 24 hour toxicity level measured.
Because the most likely human route of uptake of CYN is ingestion, oral
toxicity experiments were conducted on mice. The oral LD50 was found to be 4.4-6.9 mg CYN/kg, and in addition to some ulceration
of the oesophageal gastric mucosa
, symptoms were consistent with that of intraperitoneal dosing. Stomach contents included culture
material, which indicated that these LD50 figures might be overestimated.
changes associated with CYN poisoning were reported to be in four distinct stages: inhibition
of protein synthesis
, proliferation
of membrane
s, lipid accumulation within cells, and finally cell death. Examination of mice livers removed at autopsy showed that on intraperitoneal injection of CYN, after 16 hours ribosomes from the rough endoplasmic reticulum (rER) had detached, and at 24 hours, marked proliferation of the membrane
systems of the smooth ER and Golgi apparatus
had occurred. At 48 hours, small lipid droplets had accumulated in the cell bodies, and at 100 hours, hepatocytes in the hepatic lobules were destroyed beyond function.
The process of protein synthesis inhibition has been shown to be irreversible, however is not conclusively the method of cytotoxicity
of the compound. Froscio et al.. proposed that CYN has at least two separate modes of action: the previously reported protein synthesis inhibition, and an as-yet unclear method of causing cell death. It has been shown that cells can survive for long periods (up to 20 hours) with 90% inhibition of protein synthesis, and still maintain viability. Since CYN is cytotoxic within 16–18 hours it has been suggested that other mechanisms are the cause of cell death.
Cytochrome P450 has been implicated in the toxicity of CYN, as blocking the action of P450 reduces the toxicity of CYN. It has been proposed that an activated P450-derived metabolite
(or metabolites) of CYN is the main cause of toxicity. Shaw et al.. demonstrated that the toxin could be metabolised in vivo
, resulting in bound metabolites in the liver tissue, and that damage was more prevalent in rat hepatocytes than other cell types.
Due to the structure of CYN, which includes sulfate
, guanidine
and uracil
groups, it has been suggested that CYN acts on DNA
or RNA
. Shaw et al.. reported covalent binding of CYN or its metabolites to DNA in mice, and DNA strand breakage has also been observed. Humpage et al. also supported this, and in addition postulated that CYN (or a metabolite) acts on either the spindle or centromeres during cell division
, inducing loss of whole chromosomes.
The uracil
group of CYN has been identified as a pharmacophore
of the toxin. In two experiments, the vinylic hydrogen
atom on the uracil ring was replaced with a chlorine
atom to form 5-chlorocylindrospermopsin, and the uracil group was truncated to a carboxylic acid
, to form cylindrospermic acid (Figure 6). Both products were assessed as being non-toxic, even at 50 times the LD50 of CYN. In the previous determination of the structure of deoxycylindrospermopsin, a toxicity assessment of the compound was carried out. Mice injected intraperitoneally with four times the 5-day median lethal dose of CYN showed no toxic effects. As this compound was shown to be relatively abundant, it was concluded that this analogue
was comparatively non-toxic. Given that both CYN and epiCYN are toxic, the hydroxyl
group on the uracil bridge can be considered necessary for toxicity. As yet, the relative toxicities of CYN and epiCYN have not been compared.
Since the Palm Island outbreak, several other species of cyanobacteria have been identified as producing CYN: Anabaena bergii, Anabaena lapponica , Aphanizomenon ovalisporum, Umezakia natans, Raphidiopsis curvata. and Aphanizomenon issatschenkoi. In Australia, three main toxic cyanobacteria exist: Anabaena circinalis
, Microcystis species and C. raciborskii. Of these the latter, which produces CYN, has attracted considerable attention, not only due to the Palm Island outbreak, but also as the species is spreading to more temperate
areas. Previously, the algae was classed as only tropical, however it has recently been discovered in temperate regions of Australia, Europe
, North
and South America
, and also New Zealand
.
In August 1997, three cows and ten calves died from cylindrospermopsin poisoning
on a farm in northwest Queensland. A nearby dam containing an algal bloom was tested, and C. raciborskii was identified. Analysis by HPLC
/mass spectrometry
revealed the presence of CYN in a sample of the biomass
. An autopsy
of one of the calves reported a swollen liver
and gall bladder, along with haemorrhages of the heart
and small intestine
. Histological examination of the hepatic tissue was consistent with that reported in CYN-affected mice. This was the first report of C. raciborskii causing mortality
in animals in Australia.
The effect of a bloom of C. raciborskii on an aquaculture
pond in Townsville, Australia was assessed in 1997. The pond contained Redclaw crayfish
, along with a population of Lake Eacham
Rainbowfish
to control the excess food. Analysis revealed that the water contained both extracellular
and intracellular
CYN, and that the crayfish had accumulated this primarily in the liver but also in the muscle
tissue. Examination of the gut contents revealed cyanobacterial cells
, indicating that the crayfish had ingested
intracellular toxin. An experiment using an extract of the bloom showed that it was also possible to uptake extracellular toxin directly into the tissues. Such bioaccumulation
, particularly in the aquaculture industry, was of concern, especially when humans were the end users of the product.
The impact of cyanobacterial blooms has been assessed in economic terms. In December 1991, the world’s largest algal bloom occurred in Australia, where 1000 km of the Darling
-Barwon
River was affected. One million people-days of drinking water were lost, and the direct costs incurred totalled more than A$1.3 million. Moreover, 2000 site-days of recreation were also lost, and the economic cost was estimated at A$10 milliobn, after taking into account indirectly affected industries such as tourism
, accommodation
and transport
.
(LC-MS
), mouse bioassay, protein synthesis inhibition assay, and reverse-phase HPLC-PDA (Photo Diode Array) analysis. A cell free protein synthesis assay has been developed which appears to be comparable to HPLC-MS.
Cyanotoxin
Cyanotoxins are toxins produced by bacteria called cyanobacteria . Cyanobacteria are found almost everywhere, but particularly in lakes and in the ocean where, under certain conditions, they reproduce exponentially to form blooms. Blooming cyanobacteria can produce cyanotoxins in such...
produced by a variety of freshwater
Freshwater
Fresh water is naturally occurring water on the Earth's surface in ice sheets, ice caps, glaciers, bogs, ponds, lakes, rivers and streams, and underground as groundwater in aquifers and underground streams. Fresh water is generally characterized by having low concentrations of dissolved salts and...
cyanobacteria. CYN is a polycyclic
Polycyclic compound
In organic chemistry, a polycyclic compound is a cyclic compound with more than one hydrocarbon loop or ring structures . In general, the term includes all polycyclic aromatic compounds, including the polycyclic aromatic hydrocarbons, the heterocyclic aromatic compounds containing sulfur,...
uracil
Uracil
Uracil is one of the four nucleobases in the nucleic acid of RNA that are represented by the letters A, G, C and U. The others are adenine, cytosine, and guanine. In RNA, uracil binds to adenine via two hydrogen bonds. In DNA, the uracil nucleobase is replaced by thymine.Uracil is a common and...
derivative
Derivative (chemistry)
In chemistry, a derivative is a compound that is derived from a similar compound by some chemical or physical process. In the past it was also used to mean a compound that can be imagined to arise from another compound, if one atom is replaced with another atom or group of atoms, but modern...
containing guanidino
Guanidine
Guanidine is a crystalline compound of strong alkalinity formed by the oxidation of guanine. It is used in the manufacture of plastics and explosives. It is found in urine as a normal product of protein metabolism. The molecule was first synthesized in 1861 by the oxidative degradation of an...
and sulfate
Sulfate
In inorganic chemistry, a sulfate is a salt of sulfuric acid.-Chemical properties:...
groups. It is also zwitterion
Zwitterion
In chemistry, a zwitterion is a neutral molecule with a positive and a negative electrical charge at different locations within that molecule. Zwitterions are sometimes also called inner salts.-Examples:...
ic, making it highly water soluble. CYN is toxic
Toxicity
Toxicity is the degree to which a substance can damage a living or non-living organisms. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a substructure of the organism, such as a cell or an organ , such as the liver...
to liver
Liver
The liver is a vital organ present in vertebrates and some other animals. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemicals necessary for digestion...
and kidney
Kidney
The kidneys, organs with several functions, serve essential regulatory roles in most animals, including vertebrates and some invertebrates. They are essential in the urinary system and also serve homeostatic functions such as the regulation of electrolytes, maintenance of acid–base balance, and...
tissue and is thought to inhibit protein synthesis
Protein biosynthesis
Protein biosynthesis is the process in which cells build or manufacture proteins. The term is sometimes used to refer only to protein translation but more often it refers to a multi-step process, beginning with amino acid synthesis and transcription of nuclear DNA into messenger RNA, which is then...
and to covalently modify DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
and/or RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
. It is not known whether cylindrospermopsin is a carcinogen, but it appears to have no tumour initiating activity in mice.
CYN was first discovered after an outbreak of a mystery disease on Palm Island
Palm Island, Queensland
Palm Island is an Aboriginal community located on Great Palm Island, also called by the Aboriginal name "Bwgcolman", an island on the Great Barrier Reef in North Queensland, Australia The settlement is also known by a variety of other names including "the Mission", Palm Island Settlement or Palm...
, Queensland
Queensland
Queensland is a state of Australia, occupying the north-eastern section of the mainland continent. It is bordered by the Northern Territory, South Australia and New South Wales to the west, south-west and south respectively. To the east, Queensland is bordered by the Coral Sea and Pacific Ocean...
, Australia
Australia
Australia , officially the Commonwealth of Australia, is a country in the Southern Hemisphere comprising the mainland of the Australian continent, the island of Tasmania, and numerous smaller islands in the Indian and Pacific Oceans. It is the world's sixth-largest country by total area...
. The outbreak was traced back to a bloom
Algal bloom
An algal bloom is a rapid increase or accumulation in the population of algae in an aquatic system. Algal blooms may occur in freshwater as well as marine environments. Typically, only one or a small number of phytoplankton species are involved, and some blooms may be recognized by discoloration...
of Cylindrospermopsis raciborskii in the local drinking water supply, and the toxin was subsequently identified. Analysis of the toxin led to a proposed chemical structure
Chemical structure
A chemical structure includes molecular geometry, electronic structure and crystal structure of molecules. Molecular geometry refers to the spatial arrangement of atoms in a molecule and the chemical bonds that hold the atoms together. Molecular geometry can range from the very simple, such as...
in 1992, which was revised after synthesis
Chemical synthesis
In chemistry, chemical synthesis is purposeful execution of chemical reactions to get a product, or several products. This happens by physical and chemical manipulations usually involving one or more reactions...
was achieved in 2000. Several analogues of CYN, both toxic and non-toxic, have been isolated or synthesised.
C. raciborskii has been observed mainly in tropical areas, however has also recently been discovered in temperate regions of Australia
Australia
Australia , officially the Commonwealth of Australia, is a country in the Southern Hemisphere comprising the mainland of the Australian continent, the island of Tasmania, and numerous smaller islands in the Indian and Pacific Oceans. It is the world's sixth-largest country by total area...
, Europe
Europe
Europe is, by convention, one of the world's seven continents. Comprising the westernmost peninsula of Eurasia, Europe is generally 'divided' from Asia to its east by the watershed divides of the Ural and Caucasus Mountains, the Ural River, the Caspian and Black Seas, and the waterways connecting...
, North
North America
North America is a continent wholly within the Northern Hemisphere and almost wholly within the Western Hemisphere. It is also considered a northern subcontinent of the Americas...
and South America
South America
South America is a continent situated in the Western Hemisphere, mostly in the Southern Hemisphere, with a relatively small portion in the Northern Hemisphere. The continent is also considered a subcontinent of the Americas. It is bordered on the west by the Pacific Ocean and on the north and east...
, and New Zealand
New Zealand
New Zealand is an island country in the south-western Pacific Ocean comprising two main landmasses and numerous smaller islands. The country is situated some east of Australia across the Tasman Sea, and roughly south of the Pacific island nations of New Caledonia, Fiji, and Tonga...
.
Discovery
In 1979, 138 inhabitants of Palm IslandPalm Island, Queensland
Palm Island is an Aboriginal community located on Great Palm Island, also called by the Aboriginal name "Bwgcolman", an island on the Great Barrier Reef in North Queensland, Australia The settlement is also known by a variety of other names including "the Mission", Palm Island Settlement or Palm...
, Queensland
Queensland
Queensland is a state of Australia, occupying the north-eastern section of the mainland continent. It is bordered by the Northern Territory, South Australia and New South Wales to the west, south-west and south respectively. To the east, Queensland is bordered by the Coral Sea and Pacific Ocean...
, Australia
Australia
Australia , officially the Commonwealth of Australia, is a country in the Southern Hemisphere comprising the mainland of the Australian continent, the island of Tasmania, and numerous smaller islands in the Indian and Pacific Oceans. It is the world's sixth-largest country by total area...
, were admitted to hospital, suffering various symptoms of gastroenteritis
Gastroenteritis
Gastroenteritis is marked by severe inflammation of the gastrointestinal tract involving both the stomach and small intestine resulting in acute diarrhea and vomiting. It can be transferred by contact with contaminated food and water...
. All of these were children; in addition, 10 adults were affected but not hospitalised. Initial symptoms, including abdominal
Abdomen
In vertebrates such as mammals the abdomen constitutes the part of the body between the thorax and pelvis. The region enclosed by the abdomen is termed the abdominal cavity...
pain and vomiting
Vomiting
Vomiting is the forceful expulsion of the contents of one's stomach through the mouth and sometimes the nose...
, resembled those of hepatitis
Hepatitis
Hepatitis is a medical condition defined by the inflammation of the liver and characterized by the presence of inflammatory cells in the tissue of the organ. The name is from the Greek hepar , the root being hepat- , meaning liver, and suffix -itis, meaning "inflammation"...
; later symptoms included kidney failure and bloody diarrhoea. Urine
Urine
Urine is a typically sterile liquid by-product of the body that is secreted by the kidneys through a process called urination and excreted through the urethra. Cellular metabolism generates numerous by-products, many rich in nitrogen, that require elimination from the bloodstream...
analysis revealed high levels of proteins, ketones and sugar
Sugar
Sugar is a class of edible crystalline carbohydrates, mainly sucrose, lactose, and fructose, characterized by a sweet flavor.Sucrose in its refined form primarily comes from sugar cane and sugar beet...
in many patients, along with blood
Blood
Blood is a specialized bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells....
and urobilinogen
Urobilinogen
Urobilinogen is a colourless product of bilirubin reduction. It is formed in the intestines by bacterial action. Some urobilinogen is reabsorbed, taken up into the circulation and excreted by the kidney. This constitutes the normal "enterohepatic urobilinogen cycle".Increased amounts of bilirubin...
in lesser numbers. The urine analysis, along with faecal microscopy
Microscopy
Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...
and poison
Poison
In the context of biology, poisons are substances that can cause disturbances to organisms, usually by chemical reaction or other activity on the molecular scale, when a sufficient quantity is absorbed by an organism....
screening, could not provide a statistical link to the symptoms. All patients recovered within 4 to 26 days, and at the time there was no apparent cause for the outbreak. Initial thoughts on the cause included poor water quality and diet, however none were conclusive, and the illness was coined the “Palm Island Mystery Disease”.
At the time, it was noticed that this outbreak coincided with a severe algal bloom in the local drinking water supply, and soon after the focus turned to the dam in question. An epidemiological study of this “mystery disease” later confirmed that the Solomon Dam was implicated, as those that became ill had used water from the dam. It became apparent that a recent treatment of the algal bloom
Algal bloom
An algal bloom is a rapid increase or accumulation in the population of algae in an aquatic system. Algal blooms may occur in freshwater as well as marine environments. Typically, only one or a small number of phytoplankton species are involved, and some blooms may be recognized by discoloration...
with copper sulfate caused lysis of the algal cells, releasing a toxin
Toxin
A toxin is a poisonous substance produced within living cells or organisms; man-made substances created by artificial processes are thus excluded...
into the water.
A study of the dam revealed that periodic blooms of algae were caused predominantly by three strains of cyanobacteria: two of the genus Anabaena
Anabaena
Anabaena is a genus of filamentous cyanobacteria that exists as plankton. It is known for its nitrogen fixing abilities, and they form symbiotic relationships with certain plants, such as the mosquito fern. They are one of four genera of cyanobacteria that produce neurotoxins, which are harmful to...
, and Cylindrospermopsis raciborskii, previously unknown in Australian waters. A mouse bioassay of the three demonstrated that although the two Anabaena strains were non-toxic, C. raciborskii was highly toxic. Later isolation of the compound responsible led to the identification of the toxin cylindrospermopsin.
A later report alternatively proposed that the excess copper in the water was the cause of the disease. The excessive dosing was following the use of least-cost contractors to control the algae, who were unqualified in the field.
Structure determination
High-performance liquid chromatography
High-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
. Structure elucidation was achieved via mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
(MS) and nuclear magnetic resonance
Nuclear magnetic resonance
Nuclear magnetic resonance is a physical phenomenon in which magnetic nuclei in a magnetic field absorb and re-emit electromagnetic radiation...
(NMR) experiments, and a structure (later proven slightly incorrect) was proposed (Figure 1).
This almost-correct molecule
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...
possesses a tricyclic
Tricyclic
Tricyclics are chemical compounds that contain three interconnected rings of atoms.Many compounds have a tricyclic structure, but in pharmacology, the term has traditionally been reserved to describe heterocyclic drugs...
guanidine
Guanidine
Guanidine is a crystalline compound of strong alkalinity formed by the oxidation of guanine. It is used in the manufacture of plastics and explosives. It is found in urine as a normal product of protein metabolism. The molecule was first synthesized in 1861 by the oxidative degradation of an...
group (rings A, B & C), along with a uracil
Uracil
Uracil is one of the four nucleobases in the nucleic acid of RNA that are represented by the letters A, G, C and U. The others are adenine, cytosine, and guanine. In RNA, uracil binds to adenine via two hydrogen bonds. In DNA, the uracil nucleobase is replaced by thymine.Uracil is a common and...
ring (D). The zwitterion
Zwitterion
In chemistry, a zwitterion is a neutral molecule with a positive and a negative electrical charge at different locations within that molecule. Zwitterions are sometimes also called inner salts.-Examples:...
ic nature of the molecule makes this highly water-soluble, as the presence of charged
Charge (physics)
In physics, a charge may refer to one of many different quantities, such as the electric charge in electromagnetism or the color charge in quantum chromodynamics. Charges are associated with conserved quantum numbers.-Formal definition:...
areas within the molecule creates a dipole
Dipole
In physics, there are several kinds of dipoles:*An electric dipole is a separation of positive and negative charges. The simplest example of this is a pair of electric charges of equal magnitude but opposite sign, separated by some distance. A permanent electric dipole is called an electret.*A...
effect, suiting the polar
Chemical polarity
In chemistry, polarity refers to a separation of electric charge leading to a molecule or its chemical groups having an electric dipole or multipole moment. Polar molecules interact through dipole–dipole intermolecular forces and hydrogen bonds. Molecular polarity is dependent on the difference in...
solvent
Solvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...
. Sensitivity of key signals in the NMR spectrum
Spectrum
A spectrum is a condition that is not limited to a specific set of values but can vary infinitely within a continuum. The word saw its first scientific use within the field of optics to describe the rainbow of colors in visible light when separated using a prism; it has since been applied by...
to small changes in pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
suggested that the uracil ring exists in a keto
Keto
Keto can refer to:* The Keto people, an ethnic group of the Siberian North.* Ceto or Keto, a sea goddess in Greek mythology.* Ketone or keto group, the functional group in the chemical compounds ketones....
/enol
Enol
Enols are alkenes with a hydroxyl group affixed to one of the carbon atoms composing the double bond. Alkenes with a hydroxyl group on both sides of the double bond are called enediols. Deprotonated anions of enols are called enolates...
tautomer
Tautomer
Tautomers are isomers of organic compounds that readily interconvert by a chemical reaction called tautomerization. This reaction commonly results in the formal migration of a hydrogen atom or proton, accompanied by a switch of a single bond and adjacent double bond...
ic relationship, where a hydrogen
Hydrogen
Hydrogen is the chemical element with atomic number 1. It is represented by the symbol H. With an average atomic weight of , hydrogen is the lightest and most abundant chemical element, constituting roughly 75% of the Universe's chemical elemental mass. Stars in the main sequence are mainly...
transfer results in two distinct structures (Figure 2). It was originally proposed that a hydrogen bond
Hydrogen bond
A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. The hydrogen must be covalently bonded to another electronegative atom to create the bond...
between the uracil and guanidine groups in the enol tautomer would make this the dominant form.
Analogues
Metabolite
Metabolites are the intermediates and products of metabolism. The term metabolite is usually restricted to small molecules. A primary metabolite is directly involved in normal growth, development, and reproduction. Alcohol is an example of a primary metabolite produced in large-scale by industrial...
of C. raciborskii was identified from extracts of the algae after the observation of a frequently occurring peak accompanying that of CYN during UV
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...
and MS experiments. Analysis by MS and NMR methods concluded that this new compound was missing the oxygen
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
adjacent to the uracil ring, and was named deoxycylindrospermopsin (Figure 3).
In 1999, an epimer
Epimer
In chemistry, epimers are diastereomers that differ in configuration of only one stereogenic center. Diastereomers are a class of stereoisomers that are non-superposable, non-mirror images of one another....
of CYN, named 7-epicyclindrospermopsin (epiCYN), was also identified as a minor metabolite from Aphanizomenon ovalisporum. This occurred whilst isolating CYN from cyanobacteria taken from Lake Kinneret in Israel
Israel
The State of Israel is a parliamentary republic located in the Middle East, along the eastern shore of the Mediterranean Sea...
. The proposed structure of this molecule differed from CYN only in the orientation of the hydroxyl
Hydroxyl
A hydroxyl is a chemical group containing an oxygen atom covalently bonded with a hydrogen atom. In inorganic chemistry, the hydroxyl group is known as the hydroxide ion, and scientists and reference works generally use these different terms though they refer to the same chemical structure in...
group adjacent to the uracil ring (Figure 4).
Total synthesis
SyntheticChemical synthesis
In chemistry, chemical synthesis is purposeful execution of chemical reactions to get a product, or several products. This happens by physical and chemical manipulations usually involving one or more reactions...
approaches to CYN started with the piperidine
Piperidine
Piperidine is an organic compound with the molecular formula 5NH. This heterocyclic amine consists of a six-membered ring containing five methylene units and one nitrogen atom...
ring (A), and progressed to annulation
Annulation
Annulation in organic chemistry is a chemical reaction in which a new ring is constructed on another molecule ....
of rings B and C. The first total synthesis
Total synthesis
In organic chemistry, a total synthesis is, in principle, the complete chemical synthesis of complex organic molecules from simpler pieces, usually without the aid of biological processes. In practice, these simpler pieces are commercially available in bulk and semi-bulk quantities, and are often...
of CYN was reported in 2000 through a 20-step process.
Improvements to synthetic methods led to a revision of the stereochemistry
Stereochemistry
Stereochemistry, a subdiscipline of chemistry, involves the study of the relative spatial arrangement of atoms within molecules. An important branch of stereochemistry is the study of chiral molecules....
of CYN in 2001. A synthetic process controlling each of the six stereogenic centres of epiCYN established that the original assignments of both CYN and epiCYN were in fact a reversal of the correct structures. An alternative approach by White and Hansen supported these absolute configurations (Figure 5). At the time of this correct assignment, it was suggested that the enol form was not dominant.
Stability
One of the key factors associated with the toxicityToxicity
Toxicity is the degree to which a substance can damage a living or non-living organisms. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a substructure of the organism, such as a cell or an organ , such as the liver...
of CYN is its stability
Chemical stability
Chemical stability when used in the technical sense in chemistry, means thermodynamic stability of a chemical system.Thermodynamic stability occurs when a system is in its lowest energy state, or chemical equilibrium with its environment. This may be a dynamic equilibrium, where individual atoms...
. Although the toxin has been found to degrade
Chemical decomposition
Chemical decomposition, analysis or breakdown is the separation of a chemical compound into elements or simpler compounds. It is sometimes defined as the exact opposite of a chemical synthesis. Chemical decomposition is often an undesired chemical reaction...
rapidly in an algal extract when exposed to sunlight
Sunlight
Sunlight, in the broad sense, is the total frequency spectrum of electromagnetic radiation given off by the Sun. On Earth, sunlight is filtered through the Earth's atmosphere, and solar radiation is obvious as daylight when the Sun is above the horizon.When the direct solar radiation is not blocked...
, it is resistant to degradation by changes in pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
and temperature
Temperature
Temperature is a physical property of matter that quantitatively expresses the common notions of hot and cold. Objects of low temperature are cold, while various degrees of higher temperatures are referred to as warm or hot...
, and shows no degradation in either the pure solid form or in pure water. As a result, in turbid
Turbidity
Turbidity is the cloudiness or haziness of a fluid caused by individual particles that are generally invisible to the naked eye, similar to smoke in air. The measurement of turbidity is a key test of water quality....
and unmoving water the toxin can persist for long periods, and although boiling water will kill the algae, it may not remove the toxin.
Toxic effects
Hawkins et al.. demonstrated the toxic effects of CYN by mouse bioassay, using an extract of the original Palm Island strain. AcutelyAcute (medicine)
In medicine, an acute disease is a disease with either or both of:# a rapid onset, as in acute infection# a short course ....
poisoned mice displayed anorexia
Anorexia (symptom)
Anorexia is the decreased sensation of appetite...
, diarrhoea and gasping respiration
Breathing
Breathing is the process that moves air in and out of the lungs. Aerobic organisms require oxygen to release energy via respiration, in the form of the metabolism of energy-rich molecules such as glucose. Breathing is only one process that delivers oxygen to where it is needed in the body and...
. Autopsy results revealed haemorrhages in the lungs, livers, kidneys, small intestines and adrenal glands. Histopathology
Histopathology
Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease...
revealed dose-related necrosis
Necrosis
Necrosis is the premature death of cells in living tissue. Necrosis is caused by factors external to the cell or tissue, such as infection, toxins, or trauma. This is in contrast to apoptosis, which is a naturally occurring cause of cellular death...
of hepatocytes, lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...
accumulation, and fibrin
Fibrin
Fibrin is a fibrous, non-globular protein involved in the clotting of blood. It is a fibrillar protein that is polymerised to form a "mesh" that forms a hemostatic plug or clot over a wound site....
thrombi formation in blood vessels of the liver and lungs, along with varying epithelial cell necrosis in areas of the kidneys.
A more recent mouse bioassay of the effects of cylindrospermopsin revealed an increase in liver weight, with both lethal and non-lethal doses; in addition the livers appeared dark-coloured. Extensive necrosis of hepatocytes was visible in mice administered a lethal dose, and some localised damage was also observed in mice administered a non-lethal dose.
Toxicity
An initial estimate of the toxicity of CYN in 1985 was that an at 24 hours was 64±5 mg of freeze-dried culture/kg of mouse body weight on intraperitoneal injectionInjection (medicine)
An injection is an infusion method of putting fluid into the body, usually with a hollow needle and a syringe which is pierced through the skin to a sufficient depth for the material to be forced into the body...
. A further experiment in 1997 measured the LD50 as 52 mg/kg at 24 hours and 32 mg/kg at 7 days, however the data suggested that another toxic compound was present in the isolate of sonicated
Sonication
thumb|right|A sonicator at the [[Weizmann Institute of Science]] during sonicationSonication is the act of applying sound energy to agitate particles in a sample, for various purposes. In the laboratory, it is usually applied using an ultrasonic bath or an ultrasonic probe, colloquially known as...
cells used; predictions made by Ohtani et al. about the 24 hour toxicity were considerably higher, and it was proposed that another metabolite was present to account for the relatively low 24 hour toxicity level measured.
Because the most likely human route of uptake of CYN is ingestion, oral
Mouth
The mouth is the first portion of the alimentary canal that receives food andsaliva. The oral mucosa is the mucous membrane epithelium lining the inside of the mouth....
toxicity experiments were conducted on mice. The oral LD50 was found to be 4.4-6.9 mg CYN/kg, and in addition to some ulceration
Peptic ulcer
A peptic ulcer, also known as PUD or peptic ulcer disease, is the most common ulcer of an area of the gastrointestinal tract that is usually acidic and thus extremely painful. It is defined as mucosal erosions equal to or greater than 0.5 cm...
of the oesophageal gastric mucosa
Gastric mucosa
The gastric mucosa is the mucous membrane layer of the stomach which contains the glands and the gastric pits. In men it is about 1 mm thick and its surface is smooth, soft, and velvety...
, symptoms were consistent with that of intraperitoneal dosing. Stomach contents included culture
Culture
Culture is a term that has many different inter-related meanings. For example, in 1952, Alfred Kroeber and Clyde Kluckhohn compiled a list of 164 definitions of "culture" in Culture: A Critical Review of Concepts and Definitions...
material, which indicated that these LD50 figures might be overestimated.
Modes of action
PathologicalPathological science
Pathological science is the process in science in which "people are tricked into false results ... by subjective effects, wishful thinking or threshold interactions". The term was first used by Irving Langmuir, Nobel Prize-winning chemist, during a 1953 colloquium at the Knolls Research Laboratory...
changes associated with CYN poisoning were reported to be in four distinct stages: inhibition
Reaction inhibitor
A reaction inhibitor is a substance that decreases the rate of, or prevents, a chemical reaction.-Inhibition of a catalyst:An inhibitor can reduce the effectiveness of a catalyst in a catalysed reaction...
of protein synthesis
Protein biosynthesis
Protein biosynthesis is the process in which cells build or manufacture proteins. The term is sometimes used to refer only to protein translation but more often it refers to a multi-step process, beginning with amino acid synthesis and transcription of nuclear DNA into messenger RNA, which is then...
, proliferation
Cell growth
The term cell growth is used in the contexts of cell development and cell division . When used in the context of cell division, it refers to growth of cell populations, where one cell grows and divides to produce two "daughter cells"...
of membrane
Biological membrane
A biological membrane or biomembrane is an enclosing or separatingmembrane that acts as a selective barrier, within or around a cell. It consists of a lipid bilayer with embedded proteins that may constitute close to 50% of membrane content...
s, lipid accumulation within cells, and finally cell death. Examination of mice livers removed at autopsy showed that on intraperitoneal injection of CYN, after 16 hours ribosomes from the rough endoplasmic reticulum (rER) had detached, and at 24 hours, marked proliferation of the membrane
Biological membrane
A biological membrane or biomembrane is an enclosing or separatingmembrane that acts as a selective barrier, within or around a cell. It consists of a lipid bilayer with embedded proteins that may constitute close to 50% of membrane content...
systems of the smooth ER and Golgi apparatus
Golgi apparatus
The Golgi apparatus is an organelle found in most eukaryotic cells. It was identified in 1898 by the Italian physician Camillo Golgi, after whom the Golgi apparatus is named....
had occurred. At 48 hours, small lipid droplets had accumulated in the cell bodies, and at 100 hours, hepatocytes in the hepatic lobules were destroyed beyond function.
The process of protein synthesis inhibition has been shown to be irreversible, however is not conclusively the method of cytotoxicity
Cytotoxicity
Cytotoxicity is the quality of being toxic to cells. Examples of toxic agents are a chemical substance, an immune cell or some types of venom .-Cell physiology:...
of the compound. Froscio et al.. proposed that CYN has at least two separate modes of action: the previously reported protein synthesis inhibition, and an as-yet unclear method of causing cell death. It has been shown that cells can survive for long periods (up to 20 hours) with 90% inhibition of protein synthesis, and still maintain viability. Since CYN is cytotoxic within 16–18 hours it has been suggested that other mechanisms are the cause of cell death.
Cytochrome P450 has been implicated in the toxicity of CYN, as blocking the action of P450 reduces the toxicity of CYN. It has been proposed that an activated P450-derived metabolite
Metabolite
Metabolites are the intermediates and products of metabolism. The term metabolite is usually restricted to small molecules. A primary metabolite is directly involved in normal growth, development, and reproduction. Alcohol is an example of a primary metabolite produced in large-scale by industrial...
(or metabolites) of CYN is the main cause of toxicity. Shaw et al.. demonstrated that the toxin could be metabolised in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
, resulting in bound metabolites in the liver tissue, and that damage was more prevalent in rat hepatocytes than other cell types.
Due to the structure of CYN, which includes sulfate
Sulfate
In inorganic chemistry, a sulfate is a salt of sulfuric acid.-Chemical properties:...
, guanidine
Guanidine
Guanidine is a crystalline compound of strong alkalinity formed by the oxidation of guanine. It is used in the manufacture of plastics and explosives. It is found in urine as a normal product of protein metabolism. The molecule was first synthesized in 1861 by the oxidative degradation of an...
and uracil
Uracil
Uracil is one of the four nucleobases in the nucleic acid of RNA that are represented by the letters A, G, C and U. The others are adenine, cytosine, and guanine. In RNA, uracil binds to adenine via two hydrogen bonds. In DNA, the uracil nucleobase is replaced by thymine.Uracil is a common and...
groups, it has been suggested that CYN acts on DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
or RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
. Shaw et al.. reported covalent binding of CYN or its metabolites to DNA in mice, and DNA strand breakage has also been observed. Humpage et al. also supported this, and in addition postulated that CYN (or a metabolite) acts on either the spindle or centromeres during cell division
Cell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
, inducing loss of whole chromosomes.
The uracil
Uracil
Uracil is one of the four nucleobases in the nucleic acid of RNA that are represented by the letters A, G, C and U. The others are adenine, cytosine, and guanine. In RNA, uracil binds to adenine via two hydrogen bonds. In DNA, the uracil nucleobase is replaced by thymine.Uracil is a common and...
group of CYN has been identified as a pharmacophore
Pharmacophore
thumb|right|300px|An example of a pharmacophore model.A pharmacophore is an abstract description of molecular features which are necessary for molecular recognition of a ligand by a biological macromolecule....
of the toxin. In two experiments, the vinylic hydrogen
Hydrogen
Hydrogen is the chemical element with atomic number 1. It is represented by the symbol H. With an average atomic weight of , hydrogen is the lightest and most abundant chemical element, constituting roughly 75% of the Universe's chemical elemental mass. Stars in the main sequence are mainly...
atom on the uracil ring was replaced with a chlorine
Chlorine
Chlorine is the chemical element with atomic number 17 and symbol Cl. It is the second lightest halogen, found in the periodic table in group 17. The element forms diatomic molecules under standard conditions, called dichlorine...
atom to form 5-chlorocylindrospermopsin, and the uracil group was truncated to a carboxylic acid
Carboxylic acid
Carboxylic acids are organic acids characterized by the presence of at least one carboxyl group. The general formula of a carboxylic acid is R-COOH, where R is some monovalent functional group...
, to form cylindrospermic acid (Figure 6). Both products were assessed as being non-toxic, even at 50 times the LD50 of CYN. In the previous determination of the structure of deoxycylindrospermopsin, a toxicity assessment of the compound was carried out. Mice injected intraperitoneally with four times the 5-day median lethal dose of CYN showed no toxic effects. As this compound was shown to be relatively abundant, it was concluded that this analogue
Analog (chemistry)
In chemistry, a structural analog , also known as chemical analog or simply analog, is a compound having a structure similar to that of another one, but differing from it in respect of a certain component. It can differ in one or more atoms, functional groups, or substructures, which are replaced...
was comparatively non-toxic. Given that both CYN and epiCYN are toxic, the hydroxyl
Hydroxyl
A hydroxyl is a chemical group containing an oxygen atom covalently bonded with a hydrogen atom. In inorganic chemistry, the hydroxyl group is known as the hydroxide ion, and scientists and reference works generally use these different terms though they refer to the same chemical structure in...
group on the uracil bridge can be considered necessary for toxicity. As yet, the relative toxicities of CYN and epiCYN have not been compared.
Related toxic blooms and their impact
Anabaena circinalis
Anabaena circinalis is species of cyanobacteria of genus Anabaena. It is capable of producing saxitoxin, which is poisonous to both human and animals....
, Microcystis species and C. raciborskii. Of these the latter, which produces CYN, has attracted considerable attention, not only due to the Palm Island outbreak, but also as the species is spreading to more temperate
Temperate
In geography, temperate or tepid latitudes of the globe lie between the tropics and the polar circles. The changes in these regions between summer and winter are generally relatively moderate, rather than extreme hot or cold...
areas. Previously, the algae was classed as only tropical, however it has recently been discovered in temperate regions of Australia, Europe
Europe
Europe is, by convention, one of the world's seven continents. Comprising the westernmost peninsula of Eurasia, Europe is generally 'divided' from Asia to its east by the watershed divides of the Ural and Caucasus Mountains, the Ural River, the Caspian and Black Seas, and the waterways connecting...
, North
North America
North America is a continent wholly within the Northern Hemisphere and almost wholly within the Western Hemisphere. It is also considered a northern subcontinent of the Americas...
and South America
South America
South America is a continent situated in the Western Hemisphere, mostly in the Southern Hemisphere, with a relatively small portion in the Northern Hemisphere. The continent is also considered a subcontinent of the Americas. It is bordered on the west by the Pacific Ocean and on the north and east...
, and also New Zealand
New Zealand
New Zealand is an island country in the south-western Pacific Ocean comprising two main landmasses and numerous smaller islands. The country is situated some east of Australia across the Tasman Sea, and roughly south of the Pacific island nations of New Caledonia, Fiji, and Tonga...
.
In August 1997, three cows and ten calves died from cylindrospermopsin poisoning
Toxin
A toxin is a poisonous substance produced within living cells or organisms; man-made substances created by artificial processes are thus excluded...
on a farm in northwest Queensland. A nearby dam containing an algal bloom was tested, and C. raciborskii was identified. Analysis by HPLC
High-performance liquid chromatography
High-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
/mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
revealed the presence of CYN in a sample of the biomass
Biomass
Biomass, as a renewable energy source, is biological material from living, or recently living organisms. As an energy source, biomass can either be used directly, or converted into other energy products such as biofuel....
. An autopsy
Autopsy
An autopsy—also known as a post-mortem examination, necropsy , autopsia cadaverum, or obduction—is a highly specialized surgical procedure that consists of a thorough examination of a corpse to determine the cause and manner of death and to evaluate any disease or injury that may be present...
of one of the calves reported a swollen liver
Liver
The liver is a vital organ present in vertebrates and some other animals. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemicals necessary for digestion...
and gall bladder, along with haemorrhages of the heart
Heart
The heart is a myogenic muscular organ found in all animals with a circulatory system , that is responsible for pumping blood throughout the blood vessels by repeated, rhythmic contractions...
and small intestine
Small intestine
The small intestine is the part of the gastrointestinal tract following the stomach and followed by the large intestine, and is where much of the digestion and absorption of food takes place. In invertebrates such as worms, the terms "gastrointestinal tract" and "large intestine" are often used to...
. Histological examination of the hepatic tissue was consistent with that reported in CYN-affected mice. This was the first report of C. raciborskii causing mortality
Death
Death is the permanent termination of the biological functions that sustain a living organism. Phenomena which commonly bring about death include old age, predation, malnutrition, disease, and accidents or trauma resulting in terminal injury....
in animals in Australia.
The effect of a bloom of C. raciborskii on an aquaculture
Aquaculture
Aquaculture, also known as aquafarming, is the farming of aquatic organisms such as fish, crustaceans, molluscs and aquatic plants. Aquaculture involves cultivating freshwater and saltwater populations under controlled conditions, and can be contrasted with commercial fishing, which is the...
pond in Townsville, Australia was assessed in 1997. The pond contained Redclaw crayfish
Crayfish
Crayfish, crawfish, or crawdads – members of the superfamilies Astacoidea and Parastacoidea – are freshwater crustaceans resembling small lobsters, to which they are related...
, along with a population of Lake Eacham
Lake Eacham
Lake Eacham: is a popular lake of volcanic origin on the Atherton Tableland of Queensland, Australia, within the World Heritage listed Wet Tropics of Queensland.-Origins:...
Rainbowfish
Rainbowfish
The rainbowfish are a family of small, colourful, freshwater fish that are found in northern and eastern Australia and New Guinea and in the Southeast Asian islands....
to control the excess food. Analysis revealed that the water contained both extracellular
Extracellular
In cell biology, molecular biology and related fields, the word extracellular means "outside the cell". This space is usually taken to be outside the plasma membranes, and occupied by fluid...
and intracellular
Intracellular
Not to be confused with intercellular, meaning "between cells".In cell biology, molecular biology and related fields, the word intracellular means "inside the cell".It is used in contrast to extracellular...
CYN, and that the crayfish had accumulated this primarily in the liver but also in the muscle
Muscle
Muscle is a contractile tissue of animals and is derived from the mesodermal layer of embryonic germ cells. Muscle cells contain contractile filaments that move past each other and change the size of the cell. They are classified as skeletal, cardiac, or smooth muscles. Their function is to...
tissue. Examination of the gut contents revealed cyanobacterial cells
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
, indicating that the crayfish had ingested
Ingestion
Ingestion is the consumption of a substance by an organism. In animals, it normally is accomplished by taking in the substance through the mouth into the gastrointestinal tract, such as through eating or drinking...
intracellular toxin. An experiment using an extract of the bloom showed that it was also possible to uptake extracellular toxin directly into the tissues. Such bioaccumulation
Bioaccumulation
Bioaccumulation refers to the accumulation of substances, such as pesticides, or other organic chemicals in an organism. Bioaccumulation occurs when an organism absorbs a toxic substance at a rate greater than that at which the substance is lost...
, particularly in the aquaculture industry, was of concern, especially when humans were the end users of the product.
The impact of cyanobacterial blooms has been assessed in economic terms. In December 1991, the world’s largest algal bloom occurred in Australia, where 1000 km of the Darling
Darling River
The Darling River is the third longest river in Australia, measuring from its source in northern New South Wales to its confluence with the Murray River at Wentworth, New South Wales. Including its longest contiguous tributaries it is long, making it the longest river system in Australia.The...
-Barwon
Barwon River (New South Wales)
The Barwon River flows through New South Wales, virtually as a continuation of the Macintyre River / Border Rivers system, before joining with the Culgoa River to form the Darling River...
River was affected. One million people-days of drinking water were lost, and the direct costs incurred totalled more than A$1.3 million. Moreover, 2000 site-days of recreation were also lost, and the economic cost was estimated at A$10 milliobn, after taking into account indirectly affected industries such as tourism
Tourism
Tourism is travel for recreational, leisure or business purposes. The World Tourism Organization defines tourists as people "traveling to and staying in places outside their usual environment for not more than one consecutive year for leisure, business and other purposes".Tourism has become a...
, accommodation
Hotel
A hotel is an establishment that provides paid lodging on a short-term basis. The provision of basic accommodation, in times past, consisting only of a room with a bed, a cupboard, a small table and a washstand has largely been replaced by rooms with modern facilities, including en-suite bathrooms...
and transport
Transport
Transport or transportation is the movement of people, cattle, animals and goods from one location to another. Modes of transport include air, rail, road, water, cable, pipeline, and space. The field can be divided into infrastructure, vehicles, and operations...
.
Current methods of analysis in water samples
Current methods include liquid chromatography coupled to mass spectrometryMass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
(LC-MS
Liquid chromatography-mass spectrometry
Liquid chromatography–mass spectrometry is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high...
), mouse bioassay, protein synthesis inhibition assay, and reverse-phase HPLC-PDA (Photo Diode Array) analysis. A cell free protein synthesis assay has been developed which appears to be comparable to HPLC-MS.