WESTPAC workshop on the development of a research strategy for - - PowerPoint PPT Presentation
WESTPAC workshop on the development of a research strategy for Harmful Algal Blooms Toward the future development of HAB science in the Western Pacific What we know, and what we do not know on HABs National Research Institute of
Paralytic shellfish poisoning
H2N O
Diarrhetic shellfish poisoning
T
dinoflagellates and structure of toxins
1 3
O
H
H NH
7
N
1
+ NH2
Okadaic acids
8
Dinophysis fortii
+ H2N
9
NH OH OH
Me
N
1 2
OH O
1 1
O O Me HO
Alexandrium tamarense
O O O OH Me
Saxitoxins
Me O O OH OH Me
shellfish Amnesic
poisoning
Pseudonitzchia multiseries
HOOC CH3
Clip
CH2COOH COOH N H CH3
ドーモイ酸
H
Neurotoxic poisoning shellfish
Scallops Mussels
Azaspiracid poisoning
O H O HO H O H O Me
Gymnodinium mikimotoi
O H OH Me HO O H
NH2 COOH O
Azaspiracids
S Me H OH Me O Me OH O H H
NH
Me O H
H Me
O Me
O Me
O H O H O O H
O
H O H
Protoperidinium crassipes
H
O
O O H H H
H
O
Me
H H Me
Brevetoxins
Me
Me
Bivalves
Dinophysis spp.
OH O O R1 O HO O O O OR3 O OH O R2 OH
Dinophysis spp.
OH O O R1 O HO O O O OR3
R1 CH3 CH3 CH3
O OH O R2 OH
R2 H CH3 CH3 R3 H H acyl (palmitoyl) Okadaic acid Dinophysistoxin-1 Dinophysistoxin-3
Symptoms: diarrhoea, nausea, vomiting, abdominal pain
O Me Me O O O O O
R1 CH2OH CH3 CHO COOH C7 R R R R
O OH OH Me OH
Pectenotoxin-1 Pectenotoxin-2 Pectenotoxin-3 Pectenotoxin-6
O Me O R O O Me Me
Symptoms: ?
(by Prof. Y . Fukuyo,The University of T
Protoceratium reticulatum
OH Me R1 O
R1 H OH Y essotoxin 45-Hydroxy-yessotoxin
O Me O OH O O Me O NaO SO O O
3
Symptoms: ?
Me O Me NaO3SO O O
OH O O R2
1
R4O O O O O OR1 O O R3 OH
7
OH
R1 R2 R3 R4
dinophysistoxin-1 (DTX1) H H CH3 CH3 H CH3 H H
free toxins
dinophysistoxin-2 (DTX2) H H CH3 H dinophysistoxin-3 (DTX3) acyl CH3 CH
3
H
7-O-acyl-esters
OA diol esters R1 R2 R3 R4
1-diol-esters
OA D8 DTX4 H H CH3 CH3 H H a b
H2 C
a
OH O OSO3H H2
b
OH C OH OH O OSO3H OSO3H
CH3 OH O CH3 CH2 O O O HO O O OH O CH3 O CH3 OH CH3 CH3 OH
OH O
Shellfish enzyme
CH3 OH O CH3 CH2 O O O HO O O O O CH3 OH CH3 CH3 CH3 OH O O
OA esters (OA diolesters or 7-O acyl-OA)
CH3 OH O R2 CH2 O O
31
2
O R2O O O OCOR1 O R3
7
O CH3 OH
35
CH3 CH3 OH
0.5M NaOH/ aqueous MeOH 75C, 40 min Free OA
CH3 OH O R2 CH2 O O
31
2
O HO O O OH O R3
7
O CH3 OH
35
CH3 CH3 OH
+
O
R1COOH C16:0
HO
R2OH
PP2A inhibition potent cytotoxicity
Ca2+ Modulate the homeostasis
diarrhea, tumor promoting
human intoxication)
actin polymerization inhibition Severe damage to liver by i.p
human intoxication)
E-cadherin fragmentation
Japan (mg/kg) EU （mg/kg） CODEX (mg/kg)
：
MW(Active subunit)
OH O O R1 O HO O O O OR3 O OH O R2 OH
35 kDa
R1 CH3 R2 H CH3 CH3 R3 H H acyl (palmitoyl) Okadaic acid
Binding
Dinophysistoxin-1 CH3 Dinophysistoxin-3 CH3
O OH P OH O2N O
Hydrolysis
O OH P OH
HO O
O2N O
P OH pNPP : p-Nitrophenyl Phosphate
H× ydrolys is
pNP : p-Nitrophenol HO
O OH P OH O2N O
O2N OH
16.00 4.00 PP2A (mg/kg) 20.00 y = 1.2448x + 0.1165 R2 = 0.9744 free + esterified OA analogues 12.00 8.00 y = 0.9622x + 0.0167 R2 = 0.9905 free OAanalogues 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 LC-MS (mg/kg)
Enzymatic conversions
Protoceratium reticulatum Prorocentrum lima
National Research Institute of Fisheries Science Food Hygiene & Development research group
Dinophysis spp.
vomiting, diarrhea, abdominal pain, headache, and fever. Symptoms develop from 30 minutes to 3 hours after consumption and last for up to 4 days. Dinophysis spp and Prorocentrum spp are the causative species producing okadaic acid (OA), dinophysistoxin-1 (DTX1), dinophysistoxin-2 (DTX2) and OA diol esters. Dinophysistoxin-3 (DTX3) includes a wide range of derivatives of OA, DTX1, and DTX2, esterified with saturated and unsaturated fatty acids, products of metabolic transformations that occur in the shellfish. There are a few reports in which the presence of OA in bivalves has been associated with epibenthic dinoflagellates of the genus Prorocentrum spp. Pectenotoxins and yessotoxins are eliminated from the regulation for marine toxins on the CODEX standard.
testing of OA and DTXs. PP2A inhibition assay is useful method for testing OA and DTXs although there are few commercially available testing kits. There are commercially available certified reference materials of OA and DTXs. There have been no documented occurrences of illness to date in the Southeast Asian region, however reports of this illness can be misidentified as a bacterial or viral source and is expected to be highly under-reported.
Investigation on the cellular toxin profiles and contents of Dinophysis spp. Chemical analyses of OA and DTXs in bivalves to assess the risk of DSP in Southeast Asian region. Establish the monitoring system for DSP by LC/MS/MS or PP2A inhibition assay for protection of humane health.
Karenia breve
HO H O O H O H H O H O H H O H O H H O O O O H H H H H H O
Brevetoxin A
HO R
Karenia brevis
O O H H H H O HO H O H H O H H H O O O O H O O O H H H O O O H H O H H H H O H H O H O H H O O O O
Brevetoxin B R=
H H H H H CHO H O H N
Brevetoxin A Brevetoxin B1 R=
SO Na
3
O CH2OH NH2
Brevetoxin B2 R=
S COOH O
Lipophilic, soluble in MeOH etc. Symptom: nausea, vomiting, diarrhoea, chills, sweats, reversal of temperature, hypotension, paresthesias of lips, paralysis
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+=145 mM
Na channel
Paralytic shellfish toxins Tetrodotoxins Na+ Na+ K+=5 mM Ciguatoxins Brevetoxins
Receptor-1
Receptor-5
Na+=10 mM K+ K+=140 mM Channel
Voltage-gated sodium channel Channel close
gastrointestinal and neurological symptoms, including: tingling and numbness of the lips, tongue, and throat; muscular aches; dizziness; reversal of sensations of hot and cold; diarrhea; and vomiting. Karenia brevis is the causative species producing brevetoxins (BTXs) which is the causative toxin of NSP .
mortalities, neurotoxic shellfish poisoning (NSP). A few poisoning cases have been reported from the USA and New Zealand. BTXs are included in the regulation for marine toxins on the CODEX standard.
analytical methods for testing of brevetoxins. Official testing method for BTXs is MBA.
There have been no documented occurrences of illness to date in the Southeast Asian region, however reports of this illness can be misidentified as a bacterial or viral source or ciguatera fish poisoning (CFP).
Investigation Investigation Karlodinium. Identification
Singapore and other Southeast Asian countries. Chemical analyses of BTXs in bivalves to assess the risk of NSP Southeast Asian region. Establish the monitoring system for BTXs by LC/MS/MS MBA for
assessed as critical levels.
R1 O R3 H O HO H OH O R2 O H O HO O H R4 H
Azadinium spinosum
H O NH O O H
R1 H CH3 H R2 CH3 CH3 H R3 R4 H H H AZA1 AZA2 AZA3 H H H
R1 O R3 H O HO H
Azadinium etc.
R4
spinosum
OH O R2 O H O HO O H H H O NH O O H
R1 H CH3 H R2 CH3 CH3 H R3 R4 H H H
Lipophilic, soluble
AZA1 AZA2 AZA3 H H H
in MeOH etc. Stable under heating Symptom: diarrhoea, nausea, vomiting, abdominal pain About 40 analogues has been reported.
Figure from Mar . Drugs 2008, 6, 39-72; DOI: 10.3390/md20080004 Scanning electron micrographs of mouse small intestinal villi (A) at 8 h following a 700 μg/kg acute oral dose of AZA1 and (B) undamaged villi for comparative
, Japan.
gastrointestinal disorders including abdominal pain, nausea, vomiting, and diarrhea. Azadinium spinosum and A. poporum were species for which AZAs were reported. The toxin profile of A. spinosum consisted of AZA1 and AZA2.
combinations of AZA2, AZA11 and AZA36. Species of genus of Azadinium have so far been reported from the North Sea, the French and Irish coast of the easternAtlantic, the Mediterranean Sea, the Argentinean coast, the Korean coast, and the Chinese coast. AZAs are included in the regulation for marine toxins on the CODEX standard. LC/MS/MS is an internationally accepted analytical method for testing of AZAs.
AZAs. There have been no documented occurrences of illness to date in the Southeast Asian region, however reports of this illness can be misidentified as a bacterial or viral source.
Investigation on the cellular toxin profiles and contents of Azadinium. Chemical analyses of AZAs in bivalves to assess the risk of AZP in Southeast Asian region. Establish the monitoring system for AZAs by LC/MS for humane health care if contamination of shellfish with AZAs is assessed as critical levels.
Gambierdiscus toxicus
H H HO O H O H OH H O H H O O H H H O O H H O H H H O H O O H R1 H HO O O H H H H R2
Ciguatoxin (CTX) R1= -CH(OH)-CH2OH; R2=OH CTX 4B R1= -CH=CH2; R2=H
H H HO
Gambierdiscus toxicus
O H O H OH H O H H O O H H O H O H H O H H H O H O O H R1 H HO O O H H H H R2
Ciguatoxin (CTX) R1= -CH(OH)-CH2OH; R2=OH CTX 4B R1= -CH=CH2; R2=H
Lipophilic, soluble in MeOH etc. Stable under heating. Symptom: Gastrointestinal symptoms include nausea, vomiting, and diarrhea, usually followed by neurological symptoms such as headaches, muscle aches, paresthesia, numbness, ataxia, vertigo, and hallucinations. Reversal of temperature.
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+=145 mM
Na channel
Paralytic shellfish toxins Tetrodotoxins Na+ Na+ K+=5 mM Ciguatoxins Brevetoxins
Receptor-1
Receptor-5
Na+=10 mM K+ K+=140 mM Channel
Voltage-gated sodium channel Channel close
H H O H O O O H H O H H H H O H H O H O O O H O H H O
A
O
M
H
E
O O O H O H H H H O H O
O
CTX１B
O
52‐epi‐54‐deoxyCTX1B
M
O O
CTX4B
M
O O O O O O
CTX4A 54‐deoxyCTX1B
M M
O O H H O O H O O H H O H H O H H H O H O H H O
E
O O H H H O O H H O H H O H
CTX3C
O
poisoning for a family consisting of six persons (four women and two men) by consumption of a red snapper fish (about 5 kg body weight) caught from Phan Thiet sea (about 300 km north far from Ho Chi Minh City)
Phan Thiet
dizziness, headache, muscle and joint pain, stomach cramp, diarrhea, vomiting, and paresthesia of the extremities Neither metal taste on mouth nor inversion hot-cold feeling was recorded. A high toxicity (0.1-0.2 MU/g) was detected in the fish by the Japanese official mouse bioassay (MBA) .
Me OH H O Me HO H H O Me H O Me H O H Me Me Me O H O Me O H O O H OH H H H O Me Me H O
Gambierdiscus toxicus
H O H Me H O Me H O Me H O H O Me HO Me
Water-soluble
H O H OH HO H O H H OH O H HO H OH OH H NaO3SO OH OH OH Me OH H Me H H Me H H OH Me O OH O OH H O O H H O Me O O O O O Me O OH OSO3Na H O OH H H OH O H H H H H H H H H H HO HO OH OH OH OH H OH HO
Symptom: No information Ca2+ Maitotoxin activates permeable, non-selective cation channels, Ca2+ leading to an increase in levels of cytosolic ions.
11.0 min
LC Condition Columm Mightysil RP-18 GP φ2.0×250mm （5μm ）
gradient step 1 2 Equilibrate time(minute ） 0-10 10-20 7 95%B 20-100 100 20 Flow rate 0.3 mL/min Columm oven 30℃ Injection volume 5 μL
Retention time (min)
Mobile Phase A：W ater B：95% MeCN 50 mM HCOOH 2 mM HCOONH4
tingling of the lips and tongue, which may spread to the extremities; nausea; vomiting; diarrhea; joint pain; muscle pain; headache; reversal of sensation of hot and cold; acute sensitivity to temperature extremes; vertigo; muscular weakness; irregular heartbeat, and reduced blood pressure. Ciguatoxin (CTX)-group toxins occur in fish as a result of biotransformation of precursor gambiertoxins produced by the benthic dinoflagellate Gambierdiscus spp. They are mainly found in Pacific, Caribbean and Indian Ocean region and they are classified as Pacific (P), Caribbean (C) and Indian Ocean (I) CTX-group toxins. At present, CFP is the most common type of marine biotoxin food poisoning worldwide with an estimated number of 10 000 to 50 000 people suffering from the disease annually. Gambierdiscus toxicus isolated from T ahiti and G. polynensis isolated from Cook Islands produces CTX4A, CTX4B, CTX3C, and 49- epiCTX3C.
analogues of CTXs. Mouse bioassay (MBA) and alternative assays such as in vitro (cytotoxicity and receptor binding) assays provide sufficient detection capability and they can detect all active analogues. There are no commercially available certified reference materials of CTXs. There have been several documented occurrences of illness to date in the Southeast Asian region, however identification of the causative toxins implicated in CFP cases has not been reported. Causative toxins in CFP cases in Vietnam are CTX1B, 52-epi-54- deoxyCTX1B and 54-deoxyCTX1B (unpublished data). Although other toxins such as maitotoxin (MTX) have been isolated from G. toxicus, the other toxins have a different mode of action and have not been concluded as the causative toxins in CFP .
Investigation on the cellular toxin profiles and contents of CTXs in Gambierdiscus spp. Isolation of Gambierdiscus strains producing CTXs to prepare standard toxins for chemical analyses such as LC/MS/MS. Chemical analyses of CTXs in fish implicated in CFP cases in Southeast Asian region to identify causative toxins.
OH HO OH O O HO OH OH O OH HO OH O HO OH OH OH OH H N
2
B moiety HO OH OH A moiety OH O O O R1 OH HO OH OH OH
8
HO N H N H O
9 3
H OH OH OH HO HO OH R2 O O O R3 OH HO
R OH
4
OH O OH HO OH OH R5 OH
R1 R2 R3 R4 R5
OH
OH H OH Palytoxin Me Me H H H OH H Ostreocin-D
OH HO OH O HO OH O OH O OH HO OH O HO OH OH OH OH
The Soft coral
H N
2
B moiety
HO OH OH
A moiety
Palythoa toxica
OH O O O R1 OH HO OH OH OH
8
HO N H N H O
9 3
H OH OH OH HO HO OH R2 O O O R3 OH HO R OH
4
OH O OH HO OH OH OH R5
R1 R2 R3 R4 R5
OH
OH H OH Palytoxin Me Me H H H OH H
Water-soluble,Aq. MeOH-soluble
Ostreocin-D
Ostreopsis
causing Symptom:Rhabdomyolysis, a syndrome injuring skeletal muscle, muscle breakdown, and leakage of large quantities of intracellular (myocyte) contents into blood plasma
contaminated with palytoxins were reported in the Philippines, after consumption of the crab Demania reynaudii (1988), and in Madagascar following consumption of the sardine Herklotsichthys quadrimaculatus (clupeotoxism) (1999)
Ostreopsis ovata bloom aerosols during recreational or working activities, in Italy (2006)
Ostreopsis ovata
Ostreopsis, has an increasingly global distribution
palytoxin analogues
respiratory illnesses due to inhalation of aerosols during blooms in the Mediterranean region Dinoflagellate genus Ostreopsis spp. （O.siamensis O. ovata etc 9 species）
726.4040 (-3.5 ppm) 786.4253 (-3.0 ppm)
726.4065 744.4170
786.4276
OH HO OH
804.4382
O
80
HO OH O OH O OH HO
78
OH O HO OH
327.1935 (+4.5 ppm) 327.1920
OH OH OH H N
+2H
2
2
OH OH
(345.2026)
O HO OH O O OH HO OH OH
9
OH HO N H N O
8
H H OH OH OH
HO HO
OH OH O O O OH HO OH
309
OH O OH
2-
[M+2H-nH2O]
HO OH OH
327
HO OH OH
1277 1268 744 406 419 377 786 300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
726 804
100% 80% 60% 40% 20% 0%
Ostreopsis strains
s0743 (A) s0752 (A) KAC85 (Italy) (A) s0662 (A) s0758 (A) s0788 (A) s0579 (A-2) s0772 (B) T80624-6 (B) WK27 (B) WK51 (B) s0618 (B) s0737 (B) HF66 (B) CT4 (B) KM5 (B) s0716 (B) MB80828-3 (B) s0577 (C-1) O07421-2 (C-2) s0806 (C-3) IR33 (D-1) OU11 (D-1) s0587 (D-2)
palytoxin
not well-defined, but include myalgia and weakness, possibly accompanied by fever, nausea and vomiting. The toxicity of PlTX is strongly dependent on the route of administration; in experimental animals PlTX and ostreocin-D are much less toxic after oral than after parenteral administration. Human fatalities due to consumption of seafood suspected to be contaminated with palytoxins were reported in the Philippines, after consumption of the crab Demania reynaudii (1988), and in Madagascar following consumption of the sardine Herklotsichthys quadrimaculatus (clupeotoxism) (1999), however identification of PLTX was insufficient due to limit of detection technologies. Respiratory illness has also occurred when people were exposed to Ostreopsis ovata bloom aerosols during recreational or working activities, in Italy (2006).
the current regulatory level of PLTX in food proposed by European Food Safety Authority (EFSA) is 30 ng/g. It is known that Ostreopsis siamensis and O. ovata produces palytoxin analogues such as ovatoxins. Ostreopsis is widely distributed in worldwide including Southeast Asian region and Japan.
Investigation on the cellular toxin profiles and contents of PLTX and
Chemical analyses of PLTX and ovatoxins in shellfish to assess the risk of PLTX and ovatoxins in SoutheastAsian region.
GYM
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
SPX1 PnTX G
Step 1: 5 % B 100%B for 10 min Step 2: 100 % B for 16 min
PnTX F PnTX E 5 10 15
results in DSP mouse bioassay. There have been few documented occurrences of illness to date. Cyclic imines are still under controversial discussion to include the marine toxin regulation.
cyclic imines.
passport issues. If you want me to analyze your samples, we will help you as much as possible.