TETRODOTOXIN (TTX)POISONING

Che Nin Mana, Razak Lajis a , Samsur Mohamad b, Faazaz binti Abd. Latiffc

a) National Poison Centre, Universiti Sains Malaysia (USM), 11800 Penang, Malaysiab) Department of Aquatic Science, Faculty of Resource Science and Technology,

University Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia (UNIMAS) c) Fisheries Research Institute,Department of Fisheries of Malaysia,11960 Jalan Batu

Maung,Penang, Malaysia

TTX

Low molecular weight Zwitterionic Soluble in water Heat stable Classified as a neurotoxin

MECHANISM OF TTX

o 1ST STAGE : numbness of lips, tongue, face, hands,feets, sensations of lightness /floating. dizziness,headache, vomiting, abdominal pain, diarrhea (onset:5 mins- hours)

o 2ND STAGE : paralysis; trouble walking, swallowing, oreven sitting, breathing difficulty, cyanosis, hypotension

o 3RD STAGE : convulsions due to irregular nerve signals,respiratory failure and death (6-24 hours)

No data available for chronic symptoms

SYMPTOMS OF ACUTE POISONING

No antidote Symptomatic Treatments:1) Absorption prevention- Activated charcoal

2) Treatment- critically maintain/monitor the blood pressure &

respiration- Intubation & mechanical ventilation- Norepinephrine, dopamine-hypotension- Atropine – bradycardia- Neostigmine-Cholinesterase inhibitor- Benzodiazepine (diazepam, lorazepam) -seizure

TREATMENTS

Prognosis is good if patient survives the first 24 hours.

Babylonia japonica

© Animal-World: Courtesy Ken Childs

Tetraodon fahaka

© Biopix.dk: Jc Schou© Vibrant Sea

Hapaloclaena maculosa

Scarus ghobban

© esv-diver.at

Jania rubens

© horta.uac.pt

Dendrobates auratus

© Ron D. Holt

DISTRIBUTION OF TTX

PUFFER FISHOrder: Tetraodontiformers

Group Family Common names

1 Ostraciidae3-genus8-species

Box fishes, Cow fishes, Trunk fishes

2 Tetraodontidae13-genus37-species

Puffer fishes, Toads

3 Diodontidae3-genus6-species

Porcupine fishes

CAUSATIVE SPECIES

Order: TetraodontiformersFamlily: TetraodontidaeTetra: four and Odon: teeth

TETRAODONTIDAE IN MALAYSIA

GEOGRAPHICAL DISTRIBUTION

SOURCES OF TTX

• Exogenous

• Endogenous

Proposed mechanism of TTX accumulation in marine animals (cited from Noguchi et al., 2006)

SOURCES OF TTX - EXOGENOUS

How TTX permeate through the cell membranes which consist of highly hydrophobic lipid bilayers.

Source :Nagashima, Y., Hamada, Y., Ushio, H., Nishio, S., Shimakura, K., and Shiomi, K., Subcellular distribution of tetrodotoxin in puffer fish liver,Toxicon, 37: 1833–1837, 1999.

The subcellular distribution of TTX and its derivatives in the puffer liver : possible endogenous origin of TTX

SOURCES OF TTX - ENDOGENOUS

POISONING INCIDENCES

SabahTrengganu

Johor Sarawak

Year Case Location Source

1985 4 Sabah Med. J. Malaysia. 40 (1), 31-34.

1987 (9) Sabah Med. J. Malaysia. 42 (3), 199-200.

1997 1 Trengganu Med. J. Malaysia. 52 (2), 172-174.

2006 1 Selangor National Poison Centre

1 Sarawak National Poison Centre

2008 34 (2) Johor Med. J. Malaysia. 64 (2), 181-182

1 Sabah State Health Department Sabah

2009 6 (1) Sabah Ministry of Health, Putrajaya

5 (1) Trengganu National Poison Centre

3 (1) Sarawak Health Department, Kuching

CONSUMMED SPECIES IN MALAYSIA

Sarawak and Sabah: culture & tradition

Yellow pufferfish; Xenopterus naritus

Roes, meat, liver

Specimen Sex

Body Size Toxicity determined by mouse bioassays (MU/g)

BW (g)

TL(cm)

SL(cm)

Muscle Gonad(Testis/Ovary)

Liver

1 ♀ 350.5 25.6 22.0 <2 7.9 9.8

2 ♀ 381.5 26.0 22.0 <2 6.6 <2

3 ♂ 40.4 16.3 13.9 <2 <2 9.4

Distribution of toxicity of Xenopterus naritus,Yellow puffer from Batang Saribas, SepaohDivision.

Source: Aquatic Department, UNIMAS

PUFFER FISH FESTIVAL-OPENING CEREMONY AT BETONG, SARAWAK; 2008

PUFFER FISH FESTIVAL

1st fair: 6-7 Aug 2002 Puffer catching, selling,

preparation and eating together State Tourism calendar as an

annual event since 2004 at Manggut Village, Betong, Sarawak

Season : March till end Oct Location : along Batang Saribas

between Tg Matu (Kg Manggut) & Tg Nangka(Kg Supa)

TOXICITY VARIATIONS WITHIN & BETWEEN SPECIES

Organ distributions: Skin Roe Intestine Liver Gonad Sex Muscle

1. K. Miyazawa and T. Noguchi, J. Toxicol-Toxin Reviews. 20 (2001)11.

2. M. El-Sayed, G.A. Yacout, M. El-Samra, A. Ali and S.M. Kotb, Ecotox. Environ. Safe. 56 (2003) 367.

3. T. Noguchi, T. Takatani and O. Arakawa, J. Food Hyg. Soc. Jpn. 5(2004)146.

4. T. Noguchi, O. Arakawa and T. Takatani, Comp. Biochem. Phys.D (2006)153.

5. Y. Nagashima, et.al. Food Hyg. Saf. Sci. 51(2010) 48.

6. S. Campbell, R.M. Harada, S.V. DeFelice, P.K. Bienfang and Q.X. Li, Nat. Prod. Res. 23 (2009)1630.

Tetrodon nigroviridis

Sampadi, Kuching, Sarawak

TTX: 1.35 µg/g

Balik Pulau, Penang

TTX: 0.77 µg/g

Takifugu oblongus

1.02 µg/g

Batu Maung, Batu Maung, Batu Maung, Batu Maung,

Batu Maung, Penang Kuala Rompin, Pahang

2.20 µg/g

Liver

Muscle

TTX Levels in Various Puffer Species in Malaysian Waters

Lagocephalus sceleratus (1.71 ± 0.28 µg/g)

Xenopterus naritus (Not detected)

Lagocephalus spadiceus (Not detected)

Arothron hispidus (4.11 ± 0.32 µg/g)

Takifugu oblongus (1.38 ± .0.11 µg/g )

Tetrodon nigroviridis (1.34 ± 0.05 µg/g )

Logocephalus lunaris (3.47 ± 0.10 µg/g)

CONTRIBUTING FACTORS

accumulation of bacteria in puffers

geographical habitats

changes of season

the ecological food chain

Etc.

1. M. El-Sayed, G.A. Yacout, M. El-Samra, A. Ali and S.M. Kotb, Ecotox. Environ. Safe. 56 (2003) 367.

2. T. Noguchi, T. Takatani and O. Arakawa, J. Food Hyg. Soc. Jpn. 5(2004)146.

3. T. Noguchi, O. Arakawa and T. Takatani, Comp. Biochem. Phys. D (2006)153.

4. S. Campbell, R.M. Harada, S.V. DeFelice, P.K. Bienfang and Q.X. Li, Nat. Prod. Res. 23 (2009)1630.

5. T. Saito, T. Noguchi, Y. Shida, T. Abe and K. Hashimoto, Nippon Suisan Gakk. 57(1991)1573.

6. H.C. Jen, S.J. Lin, Y.H. Tsai, C.H. Chen, Z.C. Lin and D.F. Hwang, J.Chromatogr. B. 871 (2008) 95.

7. G.M. Yang, B.L. Bao, E. Peatman, H.R. Li, L.B. Huang and D.M. Ren, Aquaculture 262 (2007) 183

OCH3

O

OH

OH

NH

HO

HO

OH

+H2N

HN

H

11-deoxyTTX

OCH2OH

O

OH

OH

NH

OH

+H2N

HN

O

anhydroTTX

OCH2OH

O

OH

OH

NH

HO

H

OH

+H2N

HN

OH

4-epiTTX

OCH2OH

O

OH

OH

NH

HO

HO

OH

+H2N

HN

H

TTX

11-deoxyTTX

TTX

4-epiTTX

6-epiTTX

11-deoxyTTX

norTTX-6(S)-ol

norTTX-6(R)-ol

norTTX-6, 6-diol

anhydroTTX

6-epianhydroTTX

5-deoxyTTX

trideoxyTTX

OR4

O

R3

OH

NH

HO

R2

OH

+H2N

HN

R1O

R2O

R1

OH

NH

OH

+H2N

HN

O R2O

R1

OH

NH

HO

HO

O

+H2N

HN

TTX AND ANALOGUES

time [d]

0 10 20 30

inte

nsity

[%]

0

20

40

60

80

100

0

20

40

60

80

100

anhydroTTX

TTX

epiTTX

320>162

304>164

302>162

TTX and PSP Reported-Pufferfish

poisoning with saxitoxin-not TTX

Clinical presentation and symptoms are similar

Confirmatory laboratory testing must be performed

0 10 20 30

inte

nsity

[%]

20

40

60

80

100

316>298Neo

STX

300>204

dcSTX257>126

TTX

302>162anhydroTTX

320>16211-deoxyTTX

304>286

time [min]

20

40

60

80

100

National regulations

Malaysia Food act 1983

13. Food containing substances injurious to health' Any person who prepares or sells any food that has in or upon

it any substance which is poisonous, harmful or otherwiseinjurious to health commits an offence and shall be liable, on conviction, to a fine not exceeding one hundred thousand ringgit or to imprisonment for a term not exceeding ten years or to both.

13n. Food unfit for human consumption. Anyperson who prepares or sells any food that consists wholly

or in part of any portion of an animal unfit for food; commits an offence and shall be liable, on conviction, to a fine not exceeding fifty thousand ringgit or to imprisonment for a term not exceeding eight years or to both.

Food act 1983 Widespread- Public satement/

advisory-Mass media: TV, radio, news paper, official web

Surveillance –Ministry of Health

CONTROLS

NATIONAL WORKING COMMITTEE ON ISSUES RELATED TO PUFFER

27th February 2009 To reduce mortality & morbidity To identify target group To produce guidelines To allow the consumption with license

& guidelines (to sell, import & preparation) and educational outreach materials

Risk Communication on Toxic Marine Hazard (puffers)

Ministry of Health-August 2009: guideline preparations

How to identify the eatable species to avoid poisoning How to identify/differentiate the puffer from other fish

species Preparation/ cutting of puffer to avoid poisoning How to identify sign/symptom and management of

poisoning Survey on knowledge & practice related to puffer

poisoning

Risk Communication on Toxic Marine Hazard (puffers)

Survey results:– very low

knowledge among public

– 68% don’t know morphology of puffers

Proper Preparation of Puffer Fish

28%

72%

YesNo

Figure 3: Knowledge among Respondents

Training in Handling/Preparation of Puffer Fish

9%

91%

YesNo

Figure 4:

Guideline to be delivered in the form of:• Pamphlet• Poster• Website – Advisory Notes• Documentary– TV Show

GU

IDEL

INE

ON

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W T

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PR

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ERS

GU

IDEL

INE

ON

HO

W T

OC

LEA

N T

HE

PU

FFER

S

OTHER OUTREACH MATERIALS

Guide book with illustrations Monograph of puffers in Malaysia 2008 Pamphlets Posters of puffer fish species & distribution Poster on non-eatable puffers Articles in bulletin, newspapers

PUBLIC AWARENESS PROGRAMS

• Public talk• Puffer identification

KNOWLEDGE, ATTITUDE & PRACTICE CHANGE

Expand Network With Relevant Stakeholder

Introducing 24hours Emergency Line

Community Empowerment

Recommendation

Situational Analysis

Capacity Building Workshop

Formation Of Special Interest Group

Awareness Program

PU

BLI

C A

WA

REN

ESS

PR

OG

RA

MS

MONITORING FACILITIES

NO AGENCY/DEPARTMENT TOXIN TECHNIQUE

1PUBLIC HEALTH

LABORATORY, JOHOR TTXLC-MS-METHOD

DEVELOPMENT

2PUBLIC HEALTH

LABORATORY, SABAH PSP HPLC & LC-MS/MS

3 AQUATIC SCIENCE, UNIMAS TTX, PSP MOUSE BIOASSAY

4FISHERIES RESEARCH

INSTITUTE, PENANG PSP HPLC

5NATIONAL POISON CENTRE,

USM TTX GC-MS

6FISHERY DEPARTMENT,

SABAH PSP MOUSE BIOASSAY

SOURCE: FRI, PENANG

R & D in MalaysiaYear Research Agency1973 Comparative lethality of tissue extracts from

Malaysian puffer fishes Lagocephalus lunarislunaris, L. 1. spadiceus and Arothron stellatus.

UM

2006 Toxicity of the yellow puffer fish Xenopterus naritus from Sungai Saribas, Sarawak.

UNIMAS

2006 Toxicity of Puffer Fishes (Lagocephalus wheeleri Abe, Tabeta and Kitahama, 1984 and Lagocephalus sceleratus Gmelin, 1789) from the East Coast Waters Peninsular Malaysia

UKM

R & D in MalaysiaYear

2008 A hatchery to culture the yellow puffer fish FRI, Sarawak

2009

2010

Screening of tetrodotoxin in puffers using gas chromatography–mass spectrometry

Comparison of TTX levels in various puffer fishes in Malaysia

USM

USM

SUMMARY

CONTINOUS MONITORING OF TTX IN PUFFERS AND OTHER MARINE ANIMALS

PUBLIC AWARENESS PROGRAM

THANK-YOU FOR YOUR KIND ATTENTION

94 119 133 231 232 288 278 291 387

859

1280 1329

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3426

0

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4000

1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

Year

Num

ber o

f cas

esPoisoning Cases Referred to NPC

HrsHrs

2424Start 24-hours service – Nov 2003

Natural Toxins Poisonings Referred to NPC from 2006 to 2008

96 (52.17%)

31 (16.85%)25 (13.59%)

0

20

40

60

80

100

120

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Types of Natural Toxins

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