Ciguatoxins – Features, origin,
and methods for detection
Gambierdiscus toxicus
O
OO
O
O
O
O
O
O
O
O OO
R2
R1
OH
H
H
H
H
HHHO
HHHH
HH
HH
H H H
H
HH H
HO
Ciguatoxin (CTX) R1= -CH(OH)-CH2OH; R2=OH
CTX 4B R1= -CH=CH2; R2=H
H
Ciguatera Fish Poisoning (Ciguatoxins, Maitotoxins?)
Poisoning Cases
+About 20-50 thousand people suffered from the
poisoning every years by consumption of tropical and
subtropical reef fish. The most serious seafood
poisoning in terms of numbers of patients.
Ciguatera Fish Poisoning (Ciguatoxins, Maitotoxins?)
The name ciguatera was given by Don Antonio Parra in Cuba in
1787 to intoxication following ingestion of the “cigua”, the Spanish
trivial name of a sea snail, Turbo pica, reputed to cause indigestion.
The term “cigua” was transferred to an intoxication caused by the
ingestion of coral reef fishes.
More than 500 fish species have been involved in CFP.
Definition of Ciguatera Fish Poisoning
Previous definition
Seafood poisoning consisting of various symptoms
caused by consumption of tropical and subtropical reef
fish.
Recommended definition
Food Poisoning caused by ciguatoxin (CTX) analogues.
by the Food Safety Commission of Japan and EFSA
Panel on Contaminants in the Food Chain
Pacific CTXs (Type 1)
+P-CTXs consists of 13 rings.
+The E-ring consists of 7 atoms.
+CTX1B is the most potent toxin.
+CTX4A ,B are produced by Gamebierdiscus.
Structure of Pacific (P) and Caribbean (C) cituatoxins.
P-CTX3C
C-CTX1
CTXs are consisting of 13 to 14 rings. C-CTX1 is less polar than P-CTXs.
CTXs are lipophilic heat-stable molecules that remain toxic after cooking or exposure to mild
acidic or basic conditions.
P-CTX1B analogues
Indian (I) –CTX ?
Na+=145 mM
K+=5 mM
Na+=10 mM
K+=140 mM
inside
outside
cell membrane
Na+
K+
U
U
U
outside
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
Na+
Na channel
Ciguatoxins
Brevetoxins
Paralytic shellfish toxins
Tetrodotoxins
Receptor-5
Receptor-1
Voltage-gated
sodium channel
Working mechanism of Ciguatoxins
Opening of the voltage-gated sodium channel
Channel
close
Channel
open
• Gastrointestinal (e.g. vomiting, diarrhoea, nausea),
• Neurological (e.g. tingling, itching)
• Cardiovascular (e.g. hypotension, bradycardia)
• Reversal of temperature.
• Based on case reports on human intoxications it appears that a concentration of 0.01 μg P-CTX1B equivalents/kg fish is expected not to exert effects in sensitive individuals when consuming a single fish meal.
• 70 ng P-CTX1B may cause symptoms for human.
• In severe cases the symptoms may begin as soon as 30 minutes after ingestion of contaminated fish, while in milder cases they may be delayed for 24 to 48 hours.
• Fatalities is very rare but may occur due to cardiorespiratory failure.
Symptoms
Toxicity Equivalent Factors (TEFs)
Toxins Acute i.p. LD50 in mice
P-CTX1B 1
P-CTX2 0.3
P-CTX3 0.3
P-CTX3C 0.2
2,3-dihydroxy P-CTX3C 0.1
51-hydroxy P-CTX3C 1
P-CTX4A 0.1
P-CTX4B 0.05
C-CTX1 0.1
C-CTX2 0.3
Gambierdiscus is a genus of marine dinoflagellates that produce ciguatoxins, a type
of toxin that causes the foodborne illness known as ciguatera.
They are usually epiphytic on macroalgae growing on coral reefs.
The marine dinoflagellate genus Gambierdiscus occurs globally in tropic and
subtropic regions.
Currently, G. toxicus, G. polynesiensis, G. excentricus and G. silvae are recognized
as being highly toxic, but ongoing and future research might identify more toxic
compounds in other species.
Gambierdiscus toxicus
Photo from Wikipedia
P-CTXs produced by G. toxicus
Yogi et al. Anal Chem. 2011, 83
CTX3C
49-epiCTX3C
CTX4A
CTX4B
P-CTX Type 1
P-CTX Type 2
P-CTXs in several fish in Okinawa in Japan
Yogi et al. Anal Chem. 2011, 83
CTX1B
52-epi-54-
deoxyCTX1B
54-deoxy
CTX1B
• CFP has occurred frequently in Okinawa Islands.
• Ciguatoxin-1B (CTX1B), 54-deoxyCTX1B, and 52-epi-54-deoxyCTX1B have been detected in several fish (large red mumea, snapper, coral cod etc) implicated in food poisoning cases.
• CTX3C was detected in one individual coral cod (Oplegnathuspunctatus) collected in Miyazaki in Kyusyu Island.
• CFP has occurred by consumption of toxic fish meat while high toxicities were found in head and viscera.
• No fatal cases by CFP had been reported.
• CTX had not been detected in Gambierdiuscus in Japan.
• Japan has a ban on domestic sales of barracuda fish (MHWL, 1953) and several other fish species that are associated with CFP.
Japanese experiences on CFP
Analytical methods for CTXs
• Bioassays (mouse, chiken, cat, brine shrimp etc.)
• Cytotoxicity assay
• Sodium channel binding assay for CTXs
• ELISA
• LC/MS/MS
• Currently, there are no regulatory limits for CTX in fish but a guidance
level of 0.01 ppb P-CTX1B equivalent (0.1ppb C-CTX1 equivalent)
has been issued by the United States Food and Drug Administration
(US FDA).
• These recommendations were based on a 10-fold reduction of the
lowest concentration of CTX in meal remnants found to cause human
illness.
Mouse Bioassay (MBA)
• The MBA based on the method described by Banner et al. (1960) is
presently the most widely used assay for the detection of CTX-group
toxins in fish. The assay is described by Yasumoto et al. (1971, 1984)
and it has been extensively used in the Pacific area (Juranovic and
Park, 1991).
• The main advantages of the MBA are (1) the provision of a measure
of total toxicity based on the biological response of the animal to the
toxin(s), (2) it does not require complex analytical equipment.
• The main disadvantages of the MBA are (1) no specific information is
provided on individual toxins, (2) it is not sensitive enough to detect
relevant levels of CTX-group toxins, (3) it cannot be automated, (4) it
requires specialised animal facilities and expertise.
Cytotoxicity assay
• Cytotoxicity assays for the detection of CTX-group toxins in fish
tissues are based on the capacity of the toxins to bind to sodium
channels, causing them to open at normal cell resting membrane
potentials.
• This results in an influx of sodium ions, cell depolarisation and the
appearance of spontaneous action potentials in excitable cells.
• The reported cell based assay for the CTX-group toxins takes
advantage of this phenomenon to produce an assay that is highly
sensitive to CTX-group toxins.
• The LOQ of this assay for CTX-group toxins is at pg/kg shellfish level.
Sodium channel binding assay
• The assay measures the inhibition of the binding of [3H]-brevetoxin-3
to sodium-channels in rat brain synaptosomes in the presence of
CTX-group toxins.
Na+=145 mM
K+=5 mM
Na+=10 mM
K+=140 mM
inside
outside
cell membrane
Na+
K+
U
U
U
outside
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
Na+
Na channel
Ciguatoxins
Brevetoxins
Paralytic shellfish toxins
Tetrodotoxins
Receptor-5
Receptor-1
Voltage-gated
sodium channel
Working mechanism of Ciguatoxins
Opening of the voltage-gated sodium channel
Channel
close
Channel
open
Immunity to disease
Virus, toxins
etc. (antigens)
macrophage
T
Helper T cell
Transmission
B
B cell
Production of
antibody
Transmission
antibody
Detection of PSP toxins by ELISA
①二枚貝の抽出液を添加
②標識貝毒を添加
③洗浄後に発色試薬を添加
貝毒抗体を固層化したプレート
(+)
(-)
④発色率による判定
貝毒標識貝毒
標識貝毒
有毒貝
無毒貝
Toxic
sample
Non-
toxic
sample
Antibody plate
PSP
Labeled
PSP
Labeled
PSP
Antibody
Coloring
reagent
Coloring
reagent
Symptom: No information
Maitotoxin activates Ca2+ permeable, non-selective cation channels,
leading to an increase in levels of cytosolic Ca2+ ions.
Gambierdiscus toxicus
H
Me
H
Me
H
Me
H
Me
Me
O
HO
OH
OH
Me
HO
HO
OH
OH
OH
Me OSO3Na
Me
OH
MeH
H
OH
H HMe
OH
H H H H H HOH
H HO
H
O
OH H H
O
O
O
O
O
O
OH
NaO3SO OH OHH
HO
HMe H HOHO
H
H
OH
O
OH
H
O
H
OH
HH
HOH
HOH
H
Me
OHH
HO
H
H
H
Me
HHHH
H
Me
Me
Me
OH
OH
H
MeMeMeH
O
O
O
OO
O
O
O
O
H
H
H
H
H
Me
MeHO
OH
O
O O
OO
O
O
O
O
O
Maitotoxins: MW 3422Water-soluble
MTX standard (0.5 μg/mL)
11.0 min
m/z 1689.8
Retention time (min)
Extracted ion chromatogram of MTX standard obtained by qTOF/LC/MS (m/z 1000-4000)
LC Condition
Columm Mightysil RP-18 GP φ2.0×250mm (5μm)
Mobile Phase A:Water 50 mM HCOOH
B:95% MeCN 2 mM HCOONH4
gradient step time(minute) 95%B
1 0-10 20-100
2 10-20 100
Equilibrate 7 20
Flow rate 0.3 mL/min
Columm oven 30℃
Injection volume 5 μL
Strain Speacies LocationMTX concentration
(µg/mL)
MTX per cell
(pg/cell)
KW070922 G. scabrosus Kouchi ND ND
G1G G. scabrosus Okinawa ND ND
K070922-2 G. scabrosus Kouchi ND ND
M080828-3 G. scabrosus Kouchi ND ND
T080908-1 G. scabrosus Kouchi ND ND
G18G G. scabrosus Okinawa 0.07 0.02
G19G G. scabrosus Okinawa ND ND
U1G G. scabrosus Okinawa ND ND
WI29G G. scabrosus Wakayama ND ND
M080828-2 Gambierdiscus sp. Type2 Kouchi ND ND
ON2G Gambierdiscus sp. Type2 Okinawa ND ND
OI4G Gambierdiscus sp. Type2 Kouchi ND ND
OI6G Gambierdiscus sp. Type2 Kouchi ND ND
S0080911-1 G. australes Kouchi 11.17 11.39
G2G G. australes Okinawa ND ND
I080606-1 G. australes Okinawa ND ND
M080828-4 G. australes Kouchi ND ND
S4G G. australes Kouchi 19.85 17.19
U2G G. australes Okinawa 2.84 7.31
M14G G. australes Kouchi ND ND
WI11G Gambierdiscus sp. type 3 Wakayama ND ND
WI9G Gambierdiscus sp. type 3 Wakayama ND ND
Maitotoxin content of cultured Gambierdiscus sp.