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TRANSCRIPT
Marine toxin research and toxin
monitoring system in Japan:
Experiences from Japan
Toshiyuki Suzuki
National Research Institute of Fisheries
Science (NRIFS)
HN
N
O
NH
HN
HH
OH
OH
N+H2
H2+N
H2NOC
Hokkaido
Aomori
Iwate
Miyagi
Chiba
Aichi
Hiroshima
Pacific Ocean
「Kiri no hana ga sakukoroni igai wo taberuna」Don’t eat mussels during the flower season of paulownia.
(An old proverb in Japan)
Contents
Introduction of marine toxins: chemical
structure and causative algae
Japanese marine toxin monitoring system
Development of analytical methods for
marine toxins in Japan
Production of standard toxins
Introduction of marine toxins: chemical structure
and causative algae
N
NH
NH
N
R2
OH
H
R3
OH
NH2+
NH2+
R1
R4
Alexandrium spp.
Gymnodinium spp.
N-sulfo-
Carbamate carbamoyl Decarbamoyl
Toxins Toxins Toxins
R1 R2 R3 R4: OCONH2 R4: OCONHSO3- R4: OH
H H H STX GTX5 (B1) dcSTX
OH H H neoSTX GTX6 (B2) dcneoSTX
OH H OSO3- GTX1 C3 dcGTX1
H H OSO3- GTX2 C1 dcGTX2
H OSO3- H GTX3 C2 dcGTX3
OH OSO3- H GTX4 C4 dcGTX4
E.J. Schantz et al.: J. Am. Chem. Soc., 97, 1238-1239 (1975).
Y. Shimizu et al.: J. Am. Chem. Soc., 98, 5414-5416 (1976).
Y. Shimizu et al.: J. Am. Chem. Soc., 100, 6791-6793 (1978).
Paralytic Shellfish Poisoning Toxins (Saxitoxins)
O
O
O
O
OO O
OH
O
OHOR3
OH
OH R2
R1
R1 R2 R3
Okadaic acid CH3 H H
Dinophysistoxin-1 CH3 CH3 H
Dinophysistoxin-3 CH3 CH3 acyl (palmitoyl)
Me
O
O
OMe Me
OMe
OHOH
Me
O
O O
O
O
OH
Me
R
O
O
R1 C7
Pectenotoxin-1 CH2OH R
Pectenotoxin-2 CH3 R
Pectenotoxin-3 CHO R
Pectenotoxin-6 COOH R
Diarrhetic Shellfish Poisoning
Toxins (OA, DTXs)
Pectenotoxins
Dinophysis spp.
T. Yasumoto et al.: Tetrahedron, 41, 1019-1025 (1985).
Protoceratium reticulatum
O
O
O
O
O
O
O O
O O
O
NaO3SO
NaO3SO Me
Me
Me
Me
MeOH
OH
R1
R1
Yessotoxin H
45-Hydroxy-yessotoxin OH
Yessotoxins
M. Murata et al.: Tetrahedron Lett., 28, 5869-5872 (1985).
O
O
O
O
O
O
O
OO
O O
R
HO
H
H
H
H
H
O
H
HHH
HHH
HHH
Brevetoxin B R=
Brevetoxin B1 R=HN
SO3Na
O
CHO
Brevetoxin B2 R= SCOOH
CH2OH
O
NH2
OO
O
O
O
O
O
O
O
O
O
H
HO
O
H
H
H H
H
H H
H
H
H
H
H
HHH
Brevetoxin A
Karenia brevis
Y. Shimizu et al.: J. Am. Chem. Soc., 108, 514-515 (1986).
H. Ishida et al.: Tetrahedron Lett., 36, 725-728 (1995).
K. Murata et al.: Tetrahedron, 54, 735-742 (1998).
Neurotoxic Shellfish
Poisoning Toxins
(Brevetoxins)
Pseudo-nitzschia spp.
Amnestic Shellfish Poisoning
(Domoic acid)
NH
CO2H
CO2H
HO2C
H
Domoic Acid
NH
CO2H
CO2H
Kainic Acid
J.L.C. Wright et al.: Can. J. Chem., 67, 481-490 (1989)
Takeuchi et al.: Nippon Suisan Gakkaishi, 30, 237-241 (1964)
Insecticide/Tokunoshima
HOO
O
O
O
O
O
O
NH
H R2
O
H
HO
H
H
H
O
H
H
R1
R3
AZA1 H CH3 H H
AZA2 CH3 CH3 H H
AZA3 H H H H
R1 R2 R3 R4
OH
R4
Azadinium spinosum etc.
Azaspiracid Shellfish
Poisoning Toxins (Azaspiracids)
M. Satake et al.: J. Am. Chem. Soc., 120, 9967-9968 (1998).
(revised by K.C Nicolaou et al. 2004)
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 Toxins (Ciguatoxins)
Gambierdiscus toxicus
M. Murata et al.: J. Am. Chem. Soc., 112, 4380-4386 (1990).
Gambierdiscus toxicus
M. Murata et al.: J. Am. Chem. Soc., 116, 7098-7107 (1994).
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
Maitotoxin
NH
NHNH2
+
O
O
O-
HO
R2
R1
OH
OH
R1 R2
TTX OH CH2OH
6-epiTTX CH2OH OH
11-deoxyTTX OH CH3
11-oxoTTX OH CHO
11-norTTX-6(R)-ol H OH
11-norTTX-6(S)-ol OH H
Chiriquitoxin OH CH(OH)CH(NH2)COOH
R S
K. Tsuda et al.: Chem. Pharm. Bull. Jpn., 12, 1357-1374 (1964).
R.B. Woodward: Pure Appl. Chem., 9, 49-74 (1964).
M. Yotsu et al.: Tetrahedron Lett., 31, 3187-3190 (1990).
Puffer Fish Poisoning
Toxins (Tetrodotoxins)
Newt
Cynops pyrrhogaster
Flog
Blue ringed octopus
Hapalochlaena fasciata
Horseshoe crab
Marine soft corals
Palyrhoa toxica
R.E. Moore and G.J. Bartolini: J. Am. Chem. Soc., 103, 2491-2494 (1981).
D. Uemura et al.: Tetrahedron Lett., 22, 2781-2784 (1981).
Palytoxin
HO NH
NH
O O
OH
OH
OH
HO
O
HOOH
OH
OH
HO
OH
OH
OHO
O
O
HO
OH
OH
OH
HO
OH
OH
OH
HO
O
OH
HO
OH
O
OH
OH
OH
OH
OH
OH
OH
OH
HO
HO
OH
O
OHHO
OH
OH
OH
OO
O
HO
H2N
H
Ostreopsis spp.
M. Usami et al.: J. Am. Chem. Soc., 117, 5389-5390 (1995).
Ostreosin-D
HO NH
NH
O O
OH
OH
OH
HO
O
HOOH
OH
OH
HO
OH
OH
OHO
O
O
HO
OH
OH
OH
HO
OH
OH
OH
HO
O
OH
HO
OH
O
OH
OH
OH
OH
OH
OH
OH
OH
HO
HO
OH
O
OHHO
OH
OH
OH
OO
O
HO
H2N
H
Marine toxin Monitoring System in
Japan
Regulatory level of marine toxins reported by
29th CODEX Committee on Fish and Fishery
Products(CCFFP)
Biotoxins Poisoning Regulatory level
/kg
Saxitoxin group PSP 0.8 mg
Okadaic acid group DSP 0.16 ug
Domic acid group ASP 20 mg
Brevetoxin group NSP 200 MU
Azaspiracid group AZASP 0.16 ug
Japanese Shellfish Toxin Monitoring System since 1979
Bivalve monitoring Market
Consumer
HAB monitoring
Food hygiene
lawVoluntary controls
Monitoring by theofficial testing method(Mouse bioassay) Prediction of
contamination of
bivalves with PSP
and DSP toxins
Fisheries stations by
a local government
Confirming successive
three weeks under
regulatory toxicity
levels
Ciguatera fish poisoning (CFP) and palytoxin like poisoning
(PLP) in Japan between 1998 and 2008
PLP(2000, 11
patients)
PLP(2001, 2
patients)
PLP(2003, 3
patients)
×××
××
PLP(2004, 3
patients)
××PLP
(2004, 2
patients)
One
died
×
PLP
(2007, 9
patients)
CFP
(severa
l cases
in a
CFP(1998, 10
patients)
CFP(1999, 10
patients)
×
×
×
×
CFP(2006, 4
patients)
Palytoxin like poisoning
Blue humphead parrot fish Box fish Hakofugu
Scrawled filefish Box fish Umisuzume
Table 1 Edible puffer fish and the edible parts controlled by Ministry of Health, Labour
and Welfare in Japan
Parts
English name Species Muscles Skins Testes
Grass puffer Takifugu niphobles ○ - -
Finepatterned puffer Takifugu poecilonotus ○ - -
Panther puffer Takifugu pardalis ○ - -
Vermiculated puffer Takifugu snyderi ○ - ○
Genuine puffer or
Purple puffer
Takifugu porphyreus
○ - ○
*1 Takifugu obscurus ○ - ○
Red-eyed puffer Takifugu chrysops ○ - ○
Ocella puffer or
Tiger puffer
Takifugu rubripes ○ ○ ○
Eyespot puffer Takifugu chinensis ○ ○ ○
Striped puffer or
Yellowfin puffer
Takifugu xanthopterum ○ ○ ○
Spottyback puffer Takifugu stictonotus ○ - ○
Smooth-backed
blowfish
Lagocephalus inermis ○ ○ ○
Brown-backed
toadfish
Laocephalus wheeleri ○ ○ ○
Brown-backed
toadfish
Laocephalus gloveri ○ ○ ○
Slackskinned puffer Sphoeroides pachygaster ○ ○ ○
*1 Takifugu flavidus ○ - -
○: edible
- : not edible
*1: Corresponding English name is not found.
Testing methods for fish poisoning
Fish Method Regulatory
level
Puffer fish Mouse
bioassay
１０ MU/g
Ciguatera fish Mouse
bioassay
0.025 MU/g
Palytoxin like
poisoning fish
Mouse
bioassay
Development of analytical methods for marine
toxins in Japan
Development of rapid screening methods for diarrhetic
and paralytic shellfish toxins in Japan
(2003-2006)
OA PP2A assay kit
PSP ELISA kit
Instrumental methods (HPLC, LC-MS) for
confirmatory tests of screening methods
Background data
Toxin profiles of toxic plankton and bivalves
Screening assay kit vs HPLC(LC-MS), etc
ELISA assay kit for PSP toxins
PP2A inhibition assay kit for okadaic acid
LC-MS vs protein phosphatase 2A inhibition assay*1
for okadaic acid analogues
y = 0.9622x + 0.0167
R2 = 0.9905
free OA analogues
y = 1.2448x + 0.1165
R2 = 0.9744
free + esterified OA analogues
0.00
4.00
8.00
12.00
16.00
20.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
LC-MS (mg/kg)
PP
2A
(m
g/k
g)
Fluorometric HPLC method for PSP toxins by
Dr. Oshima
+
+
N
N N
H
H
N
H
N H 2
H2 N
R1
R3R2
OH
OH
R4 -O
11
12
6
1
13
Number in parenthesis: specific toxicity in MU/µmole.
STX: saxitoxin GTX: gonyautoxin dc-: decarbamoyl-
(1274)
( 33)
(1500)
(1617)
(1872)
(1080)
(160)
(180)
( 33)
( 15)
(239)
(143)
(2483)
(2295)
(2468)
( 892)
(1584)
(1803)
-
-
-
-
-
dcSTX
dcneoSTX
dcGTX1
dcGTX2
dcGTX3
dcGTX4
GTX5
GTX6
C3
C1
C2
C4
STX
neoSTX
GTX1
GTX2
GTX3
GTX4
R3
H
H
H
H
OSO3
OSO3
R2
H
H
OSO3
OSO3
H
H
R1
H
OH
OH
H
H
OH
O
C NH 2
H
NR4 :
O
CR4 : R4 : HSO3
Structures and specific toxicities of the major paralytic shellfish toxins.
GTX4 GTX1GTX5
GTX3GTX2
GTXs
STXs C-toxins
neoSTXdcSTX
STX
C1C2
Toxin
groupMobile Phase Composition
STXs2 mM heptanesulfonate in 30 mM phosphate buffer (pH 7.0)
+ 5 % CH3CN
GTXs 2 mM heptanesulfonate in 10 mM phosphate buffer (pH 7.1)
C-toxins 2 mM tetrabutyl ammonium in phosphate buffer (pH6.3)
Mobile phases (isocratic elution)
higher resolution, sharp peaks
Column: Silica based C8: longer equilibration time than
Sullivan’s polymer column
Mobile phases：
(A) 4 mM HSA in 20 mM in
PO4 buffer + 2%CH3CN
(B) 4 mM HSA in 20 mM in
PO4 buffer + 10%CH3CN0
100
8.5 10.5 20 22
Time program of mobile phases
Time (min)
(%) (B)
(A)
Column: Synergi HydroRP
(φ4.6×150 mm)
Pump: Hitachi L-2100
Flow rate: 0.8 ml/min
Detection: Excitation; 330 nm
Emission; 390 nm
40
GTX4
GTX2
GTX3
GTX5neoSTX
STX
dcSTX
GTX1
Retention time (min)
GTX1
STXneoSTX
GTX2
GTX3
GTX4
C1/2
Chromatogram of the scallop extract showing toxicity on the regulation level
Lipophilic toxins found in Japanese bivalves
O
O
O
O
OO O
OH
O
OHOR3
OH
OH R2
R1
O
O
O
O
O
O
O O
O O
O
NaO3SO
NaO3SO Me
Me
Me
Me
MeOH
OH
R1
Me
O
O
OMe Me
OMe
OHOH
Me
O
O O
O
O
OH
Me
R
O
O
R1 R2 R3
Okadaic acid CH3 H H
Dinophysistoxin-1 CH3 CH3 H
Dinophysistoxin-3 CH3 CH3 acyl (palmitoyl)
R1
Yessotoxin H
45-Hydroxy-yessotoxin OH
R1 C7
Pectenotoxin-1 CH2OH R
Pectenotoxin-2 CH3 R
Pectenotoxin-3 CHO R
Pectenotoxin-6 COOH R
7
LC-MS analysis of DSP and lipophilic toxins in Japan
Hepatopancreas
90% MeOH
（9 times volume）
Homogenize
Centrifugation
LC/MS (5µL)
OA
DTX1
DTX3
PTX1
PTX2
PTX6
YTX
Time
0 5 10 15 20 25 30
LC-MS analysis of DSP and lipophilic toxins in HP of
scallops
Time
0 5 10 15 20 25
DTX1
DTX3
PTX1
PTX2
PTX6
YTX
45-OHYTX
OA
Fragmentation diagram of okadaic acid analogues
OHO
OH
O
O
OH
O OO
OH
H
OH
OH
O
O
169
(241) 223
323 305 287
409391
787 769 751 733 715 697
[M+NH4]+
m/z 946
[M+H]+
929 911 893 875 857 839
-NH3
447429
267
-142
805
+2H
+H142
(589)-142
OA-D8
OO
O
OO
OO
O
O
OHHO
R1
OH
O
O
R2
18
34
OO
O
O
O
O
O
HO
O
OHHO
HO
O
O
R2
m/z 169
m/z 213
m/z 195
m/z 177
(m/z 371)
m/z 353
m/z 335
m/z 311
m/z 293
m/z 275
m/z 257
R1
m/z 161
m/z 473
m/z 455
m/z 437
m/z 567, 551
m/z 549, 533
m/z 531, 515
m/z 293, 309
m/z 275, 291
m/z 257, 273
+H
+H+H
+H
-H
PTX1
PTX11
R1
OH
H
R2
H
OH
[M+NH4]+
m/z 892
[M+H]+
m/z 875
m/z 857
m/z 839
m/z 821
m/z 803
-NH3
m/z 785
-H
+NH4+
(m/z 491)m/z 573
m/z 555
m/z 591
H
A
B C
DE
FG
O
O
O
O
O
O
O O
O O
O
Me
Me
Me
Me
Me
OH
HO3SO
-O3SO
80
Me
HO
-80
181 (261)
237 (317)
293 (373)
349 (429)
-80
-80
-80
405 (485)
489 (569)
855 (935)
924 (1104)
559 (639)
657 (737)
713 (793)
-80
-80
-80-80
-80
-80
-80
799 (879)-80
Fragmentation diagram of yessotoxins
Dominant toxins in bivalves in Japan
0%
20%
40%
60%
80%
100%
Sca
llop
s (
Hokka
ido
)
Sca
llop
s (
Aom
ori)
Sca
llop
s (
Iwa
te)
Sca
llop
s (
Miy
ag
i)
Blu
e M
ussels
(A
om
ori)
Blu
e M
ussels
(Iw
ate
)
Blu
e M
ussels
(M
iyag
i)
Mu
ssels
(A
kita
)
Mu
ssels
(Y
am
ag
ata
)
Mu
ssels
(N
iigata
)
To
xin
Pro
file
(%
) 45OHYTX
YTX
PTX6
PTX2
PTX1
DTX3
DTX1
Production of standard toxins
List of Shellfish Toxins Produced in Japan Food
Research Laboratories under the Standard
Toxin Distribution ProjectToxins
DSP Okadaic acid
Dinophysistoxin-1
Dinophysistoxin-3 (7-O-16:0-DTX1)
Pectenotoxin-1
Pectenotoxin-2
Pectenotoxin-6
Yessotoxin
PSP Gonyautoxin-1,4
Gonyautoxin-2,3
C1,2
Decarbamoyl-goynautoxin-2,3
Neo-saxitoxin
Japanese ongoing research project for producing reference
toxins by combining algal culture and chemical and enzymatic
reactions
National Research Institute of Fisheries Science Food Hygiene & Development research group
Prorocentrum limaProtoceratium reticulatum
OA, DTX1
PTX2
OA, DTX1YTX
Enzymatic conversions
PTX1,3,6
Dinophysis spp.
Cultivation of D. acuminata by providing Myrionecta rubra
Criptophyte
Teleaulax sp.
Ciliate
Myrionecta rubra
D. acuminata
D. fortii
Temp. 15 C
12:12h
light-dark cycle
National Research Institute of Fisheries Science Food Hygiene & Development research group
LC-MS chromatogram of DTX1 and PTX2 obtained from a culture
of D. acuminata isolated in Japan
Column: Hypersil-BDS-C8
(50 mm x 2 mm i.d)
Flow rate: 0.2 mL/min
Mobile phase: A water, B 95% MeCN
both containing 2 mM HCOONH4
and 50 mM HCOOH
Step 1: 20 % B to 100%B for 10 min
Step 2: 100 % B for 15 min
Time (min)
0 5 10 15 20 25
m/z 903.5
m/z 817.5
PTX2
DTX1
Kamiyama and Suzuki: Harmful Algae 8 (2009) 312-317
Production of toxins by D. acumianta at different temperatures
Incubation period (days)
0
20
40
60
80
100
120
140
0 10 20 30 40
OA
(ng m
L-1
)
10
14
18
22
OA
0
1
2
3
4
5
0 10 20 30 40
DT
X 1
(ng m
L-1
)
10
14
18
22
DTX1
0
50
100
150
200
250
300
350
400
450
500
0 10 20 30 40
PT
X 2
(ng m
L-1
)
10
14
18
22
PTX2
National Research Institute of Fisheries Science Food Hygiene & Development research group
Ongoing research project for producing reference toxins by
combining algal culture and chemical reactions in Japan
National Research Institute of Fisheries Science Food Hygiene & Development research group
Anabaena circinalis
C1/C2GTX2,3, dcGTX2,3
GTX5, dcSTX
GTX1,4 neoSTX
Alexandrium tamarense
Chemical
conversions
Chemical
conversions
National Research Institute of Fisheries Science Food Hygiene & Development research group
Preparation scheme of saxitoxin analogues by
chemical conversion
1% NH4HCO3 aq.
50°C, 1h
100%
1% DTTC1/C2 GTX5
1% NH4HCO3 aq.
r.t, 1h
94%
1% DTTdcGTX2
dcGTX3dcSTX0.1M KPB
pH7.0, 75°C, 1h
40%
GTX2
GTX3
0.1M HCl aq.
100°C, 5min
100%
Reported method
GTX1
GTX4 1% NH4HCO3 aq.
50°C, 1h
100%
1% DTTneoSTX
Cyanobacteria
Anabaena
circinalis
(CS-541/04)
Dinoflagellate
Alexandrium
tamarense
15 min
100%
Reported
b-MESTXChemical
Weapon
0.1M KPB
75°C, 2h
30%
Red: our method (completed)
Blue: our method (incomplated)
Contamination of Japanese bivalves
with DA, AZAs, and BTXs
NH
CO2H
CO2H
HO2C
H
Domoic Acid
Maximum level/kg of marine biotoxins in
shellfish reported by 29th CODEX Committee
on Fish and Fishery Products(CCFFP)
Biotoxins Poisoning Maximum level
Saxitoxin group PSP 0.8 mg
Okadaic acid group DSP 0.16 ug
Domic acid group ASP 20 mg
Brevetoxin group NSP 200 MU
Azaspiracid group AZASP 0.16 ug
LC conditions
Column: Hypersil-BDS-C8
(50 mm x 2.1 mm i.d)
Flow rate: 0.2 mL/min
Mobile phase: A water, B 95% MeCN
both containing 2 mM HCOONH4 and 50
mM HCOOH
Step 1: 5 % B 100%B for 10 min
Step 2: 100 % B for 10 min
LC-MS/MS chromatogram of DA, AZAs, and BTXs
Time (min)
0 5 10 15 20
DA
BTXB2
BTX3+BTXB5
AZA3
AZA1
AZA2
deoxyBTXB2
BTX2
BTX1
Time (min)
0 5 10 15 20
DA
AZA2
Mussels from A are
Time (min)
0 5 10 15 20
DA
Mussels from B area
DA DA
AZA2
LC-MS/MS chromatogram of DA and AZA2
DA AZA1 AZA2 AZA3
Positive
numbers87 8 93 4
Positive (%) 17.2 1.6 18.3 0.8
Toxin contens
(average level
ng/g meat）
2.6-213.0
(16.7)
0.06-0.23
(0.10)
0.05-2.25
(0.28)
0.03-0.04
(0.04)
Regulatory
level
（ng/g meat）
20000 160 160 160
Contamination of bivalves (n=507) with DA and AZAs
Summary
• No human poisonings due to contaminated bivalves with DSP or PSP toxins distributed on commercial markets have been reported since the establishment of the monitoring system for shellfish toxins in 1979. This indicates that the monitoring system is successful.
• Rapid screening kit for PSP and DSP was developed in Japan.
• We are going to introduce several new methods as the alternative to MBA in the toxin monitoring program in Japan, but MBA is still useful in the marine biotoxin monitoring program.
• PSP, DSP and lipophilic toxin calibration standard is available in free in Japan.
• Risk of food poisoning by DA and AZAs in Japan is extremely low, however monitoring of these toxins will be required.
Marine toxin concerns remaining
in Japan
• Ciguatera fish poisoning is spreading along the coastal
area in Japan
• The causative toxin in palytoxin like poisoning is not
clarified. Is it palytoxin?