Toxins of cyanobacteria and algae
(cyanotoxins)
• Cyanobacteria and algae
– In prevalence autothrophic organisms
– Uni-cellular and multi-cellular body
– Cyanobacteria prokaryonta
– Algae eukaryonta
• Cyanobacteria (Cyanophyta, Cyanobacteria)
– Colonies of photosyntethising cells
• Fibers or fluffs
– Prokaryotic organisms
• No nucleus, no chloroplasts nor mitochondria
• DNA in nucleoplasmatic area
• Photosynthesis similar to plants
– Classification:
• Bentic – sessile to surfaces and bed
• Planktonic – freely floating colonies
– Algal bloom
• Concentration over 10000 cells/mL
• Visible color of water
• Summer period
– Czech republic: Microcystis aeruginosa, Aphanisomenon phlos-aquae, Anabaena spp.
– Northern Europe Oscillatoria rubescens
– Some species
• Gas vacuoles
– Flotation
– Formation of foam over 106 cells/mL
– High toxicity
• Monitoring of levels in water
• Different levels of monitoring
P…pores
CS…granules of cyanobacterial starch
L…lipid droplets
PB…carboxyzomes
GV… aerotopes (gas vesicules), transversal
and longitudinal section
CY… cyanophycine granules
NP… nucleoplasma
VA… formation analogic to vacuoles (without tonoplast)
PL...plasmolema
THYL… thylacoide
R… ribosomes
TW... wall between neighboring cells in fibre
YTW… formating cell wall
W… cell wall
S… mucosa sheat
IP...invagination of plasmalema
•Obr. 1: Transversal section of cell Pseudanabaena species (Pankratz & Bowen 1963).
• Algae
– Eukaryotic organisms
– Numerous subdivisions:
• Submicroscopic variability in morphology
• Composition of photosynthetic pigments
• Composition of reserve compounds
• Cross-penetration of types
– Developmental branch
• Autotrophic organisms
• Connected with water
– Bentic or planktonic
– Frequent toxicity
• Rhodophyta red algae
• Dinophyta dinophlagelates
• Cryptophyta cryptomonads
• Chromophyta brown algae
http://vydavatelstvi.vscht.cz/knihy/uid_es-006/hesla/img__d10e3894.html
dictyosome
cell wall
root of flagella
nucleus
and nucleolus
mitochondria
endoplasmatic
reticule
flagella
plasmalema
stigma
thylacoides
pyrenoid
chloroplast
Rhodophyta (Red Algae)
• In majority marine organisms
– Only few of them in sweet water
• Lots of different structural patterns of thallus
– Unicellular microscopic
– Complex tissue-like thalluses of microscopic
parameters
– Never possess flagellate stadium
• Pigments:
– chlorophyll a and d, posses also phycobillines
(similarly to Cyanobacteria).
• Very complicated life cycles
– reproduction both non-sexual and sexual (oogamy).
• Cell surface is covered by thick polysaccharide wall
– Polysaccharides industrial materials (for example
agar).
– Food
• Marine species Porphyra - nori in Japan
• Representatives:
Corallina – very common marine species, easy incrustation
with calcite (very huge geological sediments from dead
bodies)
Batrachospermum – verticile branched sweet water species
Lemanea – similar to Batrachospermum, looks tubular, but
possess verticile branched thallus. Similar also ecology grows
epilitic (on stones) in fast flowing pure waters
Gelidium – one of the most used industrial sources of agar
Dinophyta
• Freely living flagellates
• Very complicated life cycles
– both sessile and amoeboid stadium
• Part of Dinophyta does not possess photosynthetic
apparatus and nutrition is therefore heterotrophic
– Possible active „hunt“ and phagothropia
• Pigments
– Chlorophylls a and c
• Cell surface is usually covered by huge cellulose
casing
• Very strange nucleus, called dinokaryon.
– Very big, cca 10x bigger amount of DNA, then
usual
– Chromosomes of dinokaryon are permanently
condensed and possess no histoproteins
• Dinophyta are able to produce large amounts of
toxic compounds
– Very toxic flos acquae, so called „red tide“
• Most common:
– Ceratium, Peridinium – both genera include many
species, both sweet water and marine
– Noctiluca miliaris – component of marine
luminescent plankton
Cryptophyta
• Group of small flagellates
• Pigments:
– chlorophyll a and c and fycobillines as
cyanophyta
• In addition to own nucleus:
– Organel called nucleomorph
• degenerated nucleus of endosybiont
• chloroplast
• Their surface is soft
– well digestible for different plankton
heterotrophs.
– psychrophilic.
• Representative is genus Cryptomonas
Chromophyta
• Very large division
– Includes seven classes
– Very different organisms
• microscopic diatoms
• multimeter marine seaweeds
• Identical microscopic, ultrastructural and
biochemic parameters
• Pigments
– Chlorophyll a and c , mostly also xantophyll
fucoxanthine
– Chloroplasts 4 membranes
• Two of them are membranes of endoplasmatic
reticule, mostly connected with nucleus
• Under surface of chloroplast wreath-like
lamela – showing position of chloroplast
DNA
• thylacoids are connected in triplicates
• Storage compound is chrysolaminaran,
deposited outside of chloroplast, never
starch (further storage compounds: oils,
polyphosphate grains – volutine and others)
• Flagellate stadia possess two heteroconte
(non-comparable)) flagella, which are
different in longitude, function snd strucutre
of mastigonemates
Classification of Cyanobacterial and Algal Toxins
• Neurotoxins and paralytic poisons
• Hepatotoxins
– alkaloid
– peptidic
• Tumor promoting factors, genotoxines and mutagens
• Cytotoxins, prymnetoxins
• Embryotoxins
• Dermatotoxic alkaloids
• Lipopolysaccharides
• Immunotoxins and alergens
• Mixed bioactivity common
• Population of one species produces more different toxins
Neurotoxins and paralytic poisons
(Paralytic shellfish poisons)
• Representative compounds:
– anatoxin a, anatoxin a(s), anatoxin b,
homoanatoxin
– saxitoxin, neosaxitoxin
– aphantoxins 1-5
– gonyautoxins
– Chemical structure:
• Purine derivatives
– Saxitoxins, aphantoxins,
gonyautoxins
– Tricyclic perhydropurine
– Different substitution
• Derivatives of cyclic N-
hydroxyguanine
– Anatoxin a(s)
• Simple bicycles
– Anatoxin a, homoanatoxin a
BRIEF REVIEW OF NATURAL NONPROTEIN NEUROTOXINS Jiri Patocka and Ladislav Stredab
Sources:
• Gonyaulax Dinophyta
– Marine algae
• Anabaena, Aphanizomenon
– Cyanobacteria
• Principles of effect:
– Aphantoxins, saxitotin, neosaxitoxin – blockade
of transfer of neural excitements via blocking of
Na+ channels. No influence on K+ channels
– Anatoxin A and homoanatoxin causes change of
function in preganglial neural terminations,
acetylcholine receptors, increases the flow of
Ca2+ ions into cholinergic neural terminations
– Anatoxin a(s) acts as blocker of cholinesterase,
causes depolarisation of postsynaptic
terminations, affects nicotinic, muscarinic and
acetylcholine receptors
– Saxitoxin is je blocker of Na+ channels (first
toxin with essential influence for explanation of
Na+ and K+ channels function and neurobiology),
tetrodotoxin disrupts action potential of neural and
muscular fibers
• Symptoms of intoxication by
anatoxins
– Anatoxin-a, homoanatoxin-a,
anatoxin-a(s)
• Anabaena flos-aquae
• postsynaptic depolarizing
neuromuscular blockers
• inhibitors of
acetylcholinesterase
• Strong interaction with
nicotine receptor
• Hypersalivation
• Diarrhea
• Paralysis
• Death caused by respiratory
failure
• Potential war poisons (chemical
warfare)
– Absorption
• Inhalation
• Intact skin
• Per oral
• Introduction into food
chain
– Accumulation in
Crustaceans and fishes
– Both dependent and
independent on climate
• Intoxication PSP
– Relaxation of smooth
muscles
– Depression of action
potential in heart
– Block of sodium channel
• Guanidine ring condition
of effect
• Block from outer side of
channel
• Blocked both open and
closed channel
http://www.pac.dfo-mpo.gc.ca/ops/fm/shellfish/Biotoxins/closures/default_e.htm
•Cysts contain possible 1000 times higher amounts of toxines
• Symptoms of saxitoxin intoxication
– Consumption of contaminated food
• Oysters, Crustaceans
• Very rapid onset
– LD p.o. 0.5 mg, i.v. 0.05 mg
– Anesthesia and immobility of tongue and fingers
– Sense of thirst
– Pain in tips of fingers
– Massive intoxication
• GIT disorders
• Headache
• Disorder of movement coordination
• Ascendant type of paralysis
• Disorders of cognitive functions
• Respiratory paralysis
• For differential diagnostics absence of hypotension
• PSP compounds
– saxitoxin, neosaxitoxin, gonyautoxin I, gonyautoxin III, and decarbamoyl saxitoxin
• Toxicity similar
– gonyautoxins II, IV, V, VI, VIII, VIII-epimer, sulphocarbamoyl gonyautoxin I, IV
• Substantially less toxic
– Toxicity strongly dose-dependent
• Usage
– Chemical warfare
– Experimental compounds
Tetrodoxin TTX
– Potent and rapid action
– Tetraodontiformes
• tetraodon, pufferfish
• ovaria, liver, guts highest content
• skin traces only
• In Japan 646 of cases between 1974 nda 1983
(179 mortal), in present time 30-100 per year
– Some frogs, octopuses, snails and slugs
– Unusual tricyclic structure
• guanidinium toxins
• aminoperhydroquinazoline
– Specific blocker of Na+ channels of neurons
– Tetrodotoxin – Na+ binding site
extremely narrow
– TTX acts as hydrated Na+
– Inters the channel orifice, binding to
a glutamate residue in channel
peptide
– Conformation changes
– Electrostatic binding to an open
channels
http://www.life.umd.edu/grad/mlfsc/zctsim/ionchannel.html
– Extreme toxicity TTX
• Minimal p.o. is 30 μg/kg
– Decomposition in acidic
environment in stomach
• Termostable, decomposition in acids and
bases
– Symptoms of intoxication
• In minutes or hours
• Trembling, tingling and paresthesia of
tongue, lips and tips of finger
• Headache, nausea, vomiting, diarrhea
• Second degree
– Continuous paresthesia
– Paralysis
– Impossibility of movements
– Convulsions, arrhythmia, mental
confusion
• Death caused by respiratory arrest aprox.
In 8 hours
– Possible full consiousness close
before death
Brevetoxins
•Gymnodinium breve (Ptychodiscus
brevis)
–So called red tide
•Massive death of fishes
•Mexican gulf, Australia, coast of N.
America
–Polycyclic ethers
•Lipophilic
•10 and 11 rings
•All-trans arrangement
•Relatively stable compounds (only very
high and very low pH can cause
decomposition)
–Mechanism
•Depolarisation, opening ofotevření
napěťově řízených Na+ kanálů
•Uncontrolled influx of Na+ into cell
•Change of voltage necessary for
channels opening, hyperexcitability
–Symptoms:
•Often confused with ciguatoxins
intoxication
•Tingling of face, throath, fingers
•Trembling, nausea, vomiting, diarrhea,
headache
•Mydriasis
•Slowering of heart rate
•No mortal cases described
•Ciguatera toxins
–Mixture of compounds
•In present time 24 relative
compounds (ciguatoxin,
maitotoxin, scaritoxin, okadaic
acid)
–Dinoflagellate Gambierdiscus
toxicus
•Corall reefs
–Found in tropical fish
•Tropics and subtropics
–Low molecular lipid polyethers
–Resistant to temperature
–Stimulation of Na+ transition
through
membrane
–Neurotoxins
–4 categories of symptoms
•Neurologic - 7 days
•Cardiovascular
•Gastrointestinal 1-2 days
•General 1-7 days
– Onset of intoxication:
• 10 minutes to 12 hours after contact,
after intake of contaminated fishes
possible 36 hours
– Beginning of poisoning
• Vomiting, general weakness, diarrhea
• Decreased sensitivity to painful
stimules
• Tingling and burning of fingers
• Sense of changing cold and heat
– Further stadia
• Hypotension, mydriasis, arrhythmia
• Convulsions, circulatory collapse,
respiratory failure, death
– Possibility of persistence of symptoms
(observed for months and years)
– Difficult diagnostic from other NSP
– First aid
• Mannitol – diuretic
• Control of life functions
• No antidote
• Treatment of long-termed symptoms
– Amitriptiline, gabapentine
Domoic acid
– Nitzchia pungens
– Amnesic shellfish poisoning (ASP)
• Intoxication accompanied by neurologic disorders
– Hallucination, time-space disorientation
– Loss of short-term memory
– Symptoms of intoxication
• Vomiting, stomach convulsions, diarrhea, headache
• ASP
– Accumulation of toxin in hepatopancreas, branchiae, so
called siphon of pelecypods
– Pelecypods resistant, meat becomes toxic
– New Zealand, coast of Canada, Mexico
– Red tide
– Structure:
• Tricarboxylic acid
• Derivative of proline
• Structural similarity with excitation aminoacids (cainate,
glutamate)
– Mechanism of effect:
• Excitation AMA
• 100times higher effect then glutamate
– Rigidity of ring
• Binding to a NMDA receptor
– Influence on Ca2+ channels, entry of calcium into cell
» Stimulation of many proceses → damage of neurons
– Mediation of loss of memory
http://www.regione.emilia-romagna.it/laguna/immagine_dettaglio.asp?id_img=1002
– Doses:
• 0.9-1.9 mg/kg GIT disorders
• 1.9-4.2 mg/kg neurotoxic to lethal
– Clinical symptoms:
• neurotoxic symptoms predominating
– Headache, vertigo, confusion, time-space distortions
– Disorders of motoric coordination, hallucinations, loss of short
termed memory
• gastrointestinal difficulties
• excessive secretion of mucus into respiratory tract
• tachycardia, peripheral vasodilatation and hypotension
• cardiac arrhythmia and coma.
• Intoxication can terminated sudden death 12 to 14 hours
caused by respiratory paralysis
– Therapy:
• antagonists of NMDA
• prophylactic administration of melatonin
Hepatotoxins
• Representatives of compounds
– Alkaloid:
• Cylindrospermopsine
– Protein:
• Microcystins (cyanoginosin) and nodularins
– cyclic heptapeptides
• Sources:
– Trichodesmium, Umezakia, Cylindrospermopsis, Aphanizomenon, Microcystis,
Anabaena, Planktothrix, Nostoc, Anabaenopsis, Nodularia
• Mechanism of action:
– Inhibitors of proteosynthesis and synthesis of glutathione
– Active inhibitor of eukaryota protein serine/threonine phosphatases 1 and 2A.
• Uncontrolled phosphorylation of target proteins leads to cell proliferation, posttranslational modification of
proteins, errorneous transmission of signals nad to cellular transformation to a cancer type cell
• Cylindospermopsine
– Cylindrospermopsis raciborskii,
Umezakia natans, Aphanizomenon
ovalisporum
– Alkaloid
• tricyclic guanidine derivative bridged
with hydroxymethyluracil
• OH group of the bridge necessary for
toxicity
– Liver damage
• Inhibition of proteosynthesis,
proliferation of smooth
endoplasmatic reticulum
• Fat degeneration
• Centrilobular necrosis
– Common in tropical waters,
especially Australia
– Health problems:
• Water contamination, strong need of
purification
• Degradation products also toxic
• Mycrocystins (cyanoginosins)
– Seven AMA
– To date more than 50 compounds known
• Nodularins
– 5 AMA
– amino-9-methoxy-10-fenyl-2,6,8 trimethyl deca-
4,6-dienooic acid (ADDA) essential for
pharmocologic/toxic activity
– Mechanism of effect
• Hepatotoxicity
• Inhibitors of serine/threonine protein phosphateses
– Inhibition of dephosphorylation, switchers of
function
• Good penetration into liver cells
– High concentration, extracted together with bile acids
– Influence of cytoskeletar filaments reparation
» Collapse of cytoskelet
» Implosion of cell
» If cells neighboring with vein – penetration of
blood to liver tissue, gathering of blood in
liver
» Inflammation, death
• Symptoms of intoxication
– Vomiting, nausea
– Pale skin, cyanosis, breath difficulties, hepatic coma,
death
• Promotors of cancerogenesis of liver
– Epidemiological studies in China
– Brasil
– Contamination of drinkable water
• In danger both human and animals
http://www.abo.fi/~jmeriluo/Resources/structure.gif
Tumor Promoting Factors, Genotoxins and Mutagenes
• Compounds representatives:
– Microcystins, nodularin
– Okadaiic acid
– Sources:
• Microcystis, Nodularia
• Principples of effect:
– Inhibitors of proteinphosphatases (PP) type 1, 2A and 3
– Elevated activity of phosphorylation of proteins caused by
inhibition of proteinphosphatases
– Uncontrolled phosphorylation of target proteins lead to cellular
proliferation, posttranslational modification of proteins, errorneous
signal transmission and cell transformation to a cancer cell type.
• Nodularin more active than microcystins
– Nodularin not only tumor promoting factor, but can also initiate.
• Lower molecular weight – higher penetration to hepatocytes
• Promotors of liver cancerogenesis
– Epidemiologic studies in China
– Brazil
Cytotoxins, prymnetoxins
• Compounds representatives:
– Tubercidine
– Proteolipids
– Macrolides
• Amphidinolid B
• Caribenolid
• Goniodomin
– Polycyclic ethers
• Prymnesins
• Mechanism of primnetoxins effect:
– Decay of blood elements
– Cytotoxicity - integrity of cell membranes
– Ichtyotoxicity (jen na žábry - obojživelník po metamorfóze není intoxikován, zatím co pulec umírá do 5 min)
• Cytotoxic and cytostatic effects
– Biotechnologic promising organisms
– Freshly isolated species (from natural enviroment) higher producing ability than „pure“ laboratory species
• Cyanobacteria Spirulina subsalsa
• Alga Chlorella pyrenoidosa
– Cytostatics stopping development of S-180 cancer type
Dermatotoxic compounds
• Lyngbyatoxins A, B, C
– Lyngbya majuscula
• Bentic marine cyanobacteria
• Ebromoaplysiatoxin
– Proteinkinase C activator
– Dermatitis
– Puchýřnatění pokožky
– Tumor promotor
Lipopolysaccharides
• Mechanism of effect:
– Increased capillary permeability for proteins, effect on nonspecific
immune response, part of LPS complex so-called Oantigenic
region posses several antigenic dominants with
receptor site for lysogenic bacteriophages.
– Chemical composition of LPS is not very different fo cell
wall of Salmonella species.
– Difficult to predict physiologic activity of single species
without performance of tests
• Symptoms
– Pustules, nausea, vomiting, diarrhea
Imunotoxiny a alergeny
• Vodnatá rýma, ekzémy, slzení očí, spasmus
bronchů
• Kosmopolitní organismy
– Žijí všude, identifikovány i domácím prachu
• Vysoký obsah proteinů
• Počet alergiků vzrůstá
– ?změna alergenů nebo zvýšená citlivost?
– Přesun od vláknitých ke koloniálním sinicím
– Narůstající kontaminace stojatých vod