Toxicity of puffer fish: two species (Lagocephalus laevigatus, linaeus 1766 and Sphoeroides spengleri, Bloch 1785) from the Southeastern Brazilian coast

ORIGINAL PAPER

Toxicity of puffer fish - two species (Lagocephalus laevigatus, linaeus 1766 and Sphoeroides spengleri, Bloch 1785) from the Southeastern Brazilian coast

J. S. Oliveira^I; O. R. Pires Junior^II; R. A. V. Morales^{II, III}; C. Bloch Junior^III; C. A. Schwartz^II; J. C. Freitas^I

^IDepartment of Physiology, Institute of Biosciences and Marine Biology Center, University of São Paulo, São Paulo, Brazil

^IILaboratory of Toxinology, Department of Physiological Sciences, University of Brasilia, DF, Brazil

^IIINational Center of Genetic Resources and Biotechnology, Cenargen, Embrapa, DF, Brazil

^{Address to correspondence}

ABSTRACT

In Brazil, where puffer fish are considered poisonous, there are few documented cases on human consumption and consequent poisoning. In this study, toxicity of two puffer fish species from the Brazilian coast was examined. Specimens of Sphoeroides spengleri and Lagocephalus laevigatus were caught in São Sebastião Channel (North coast of São Paulo State, Brazil) between January 1996 and May 1997. Acidic ethanol extracts from muscle and skin plus viscera were tested for mice acute toxicity using the standard method of Kawabata. Polar extracts of S. spengleri showed high toxicity up to 946 MU/g. Extracts from L. laevigatus showed very low levels of toxicity, never exceeding 1.7 MU/g. All extracts from both species blocked amielinic nerve fiber evoked impulses of crustacean legs; this effect reverted on washing similar to the standard tetrodotoxin TTX. The aqueous extract solutions were partially purified using an ionic exchange column (Amberlit GC-50) followed by treatment with activated charcoal (Norit-A). The presence of TTX and their analogs in the semi-purified extracts were confirmed by HPLC and mass spectrometry (MALDI-TOF).

Keywords: puffer fish, Brazilian puffer, tetrodotoxin (TTX), envenoming, HPLC and mass spectrometry.

INTRODUCTION

Puffer fish are considered poisonous because they possess a potent neurotoxin, tetrodotoxin (TTX) and its analogs, which act on site 1 of the voltage-dependent sodium channels of excitable membranes, blocking sodium influx and, consequently, action potential (3). These toxins are acquired through the food chain or from symbiotic bacterial strains found on skin or in the digestive tract of these fish (6,18,24,29,32). In some localities, puffer fish species can be utilized for food. Food poisoning by human consumption of toxic puffers has occurred, especially in Japan and China where the flesh of these fish is considered a delicacy (8).

Lagocephalus laevigatus, a species found in the Atlantic Coast of the United States, showed no considerable toxicity (15). However, geographic distribution, seasonal and individual variation may produce different toxicity levels in puffers (17). Some species of puffers belonging to the Sphoeroides genus, such as S. dorsalis, S. maculatus, S. spengleri, and S. testudineus are generally considered poisonous, but in some cases, their toxicity level is not completely known (2,4,16,17).

In Brazil, there are at least nine species of puffer fish belonging to the Tetraodontidae family. They are sometimes found in local coastal city markets. There are few records of people suffering from food poisoning, possibly by consuming specimens of the Sphoeroides genus (1). There is sporadic consumption of L. laevigatus flesh (muscle and liver) around the São Sebastião Channel area. Up until now, there are no cases of food poisoning due to consumption of this species in São Paulo State. On the other hand, S. spengleri is occasionally caught but always discarded by fishermen, who consider it a very poisonous species. In this work, we investigated the toxicity level and identified the main toxic compounds present in extracts from the flesh and skin plus viscera of L. laevigatus and S. spengleri, two common species found in São Paulo coastal waters.

MATERIALS AND METHODS

Eighty-five specimens of Lagocephalus laevigatus, Linaeus, 1766 (25.0 to 52.0 cm in length and weighing up to 1.3 kg) and eighty-five Sphoeroides spengleri, Bloch, 1785 (± 15.0m in length and 30.0g) were caught at different localities in the São Sebastião Channel, at North Coast of São Paulo State, between January 1996 and May 1997 (Figure 1). The specimens were transported alive in a Styrofoam box to the laboratory, dissected, and submitted to toxin extraction procedures. For the mouse bioassay tests, the extraction procedure was according to Sato et al. (22). Briefly, alcoholic extracts (75% CH₃CH₂OH: 1% CH₃COOH; 1 mL/g) were obtained from muscle, and separately, from skin plus viscera in both species. The extracts were filtered, vacuum concentrated, defatted with methylene chloride (80% CH₂Cl₂: 20% H₂0), and polar fractions were diluted in a known concentration. One milliliter of polar fractions was intraperitonially administered in male Swiss Webster white mice (12-22g). The lethal potency was calculated from Kawabata’s table for dose-death time relationship for TTX, which defines 1 mouse unit (MU = 0.22mg of TTX) as a dose that kills a 20g male mouse within 30 minutes (15). Data were plotted as MU/g of fresh tissue (taken from 5 to 10 mice for each sample) and shown as mean ± standard error of the mean (S.E.M.).

Figure 1.
Collecting sites of Lagocephalus laevigatus (L) and Sphoeroides spengleri (S), two common puffer fish from the Southeastern Brazilian coast

For the chromatography and mass spectrometry, aqueous extracts of muscle and skin plus viscera from L. laevigatus and S. spengleri were added to an ionic exchange column (Amberlite GC-50 1x3 cm - ammonium form), vigorously washed, and eluted with 100 mL of 10% acetic acid solution. The eluted material was neutralized, concentrated, and treated with activated charcoal (Norit-A). The resulting adsorbed toxins were eluted with an ethanolic acid solution (1% Acetic Acid +20% ethanol), evaporated under reduced pressure, and re-suspended in deionized water. Samples of semi-purified extracts with 2MU equivalent toxicity were injected in a FLD-HPLC system, as per conditions to Shimpack CLC-ODS (0.46 x 250 mm) column; the mobile phase was 0.06N heptafluorobutiric acid (HFBA), 0.001N ammonium acetate (pH 5.0), and 0.5 mL/min flow rate (modified from 31). A post-column reaction with 4N NaOH at 120°C generated a C-9 base (2-amino-6-hydroximethil-8-hydroxiquinazoline), which was monitored by a fluorimeter with excitation at 375 nm and emission at 500 nm (28).

Tetrodonic acid (TDA), 4-epitetrodotoxin (4-epiTTX), and anhydrotetrodotoxin (anhydro-TTX) were synthesized according to the method developed by Mosher (19); pure TTX was purchased from Sigma-Aldrich Company Inc.

Toxins were identified by retention time comparison to standard toxin. Relationship between standard TTX applied to the FLD-HPLC and its peak area was examined in triplicate for eight concentrations over the range of 0.055 to 7.4mg. The calibration curve obtained showed a good linear correlation (R²=0.9936). The collected peaks without post-column derivatization were submitted to spectrometry analysis using a MALDI-TOF Voyager-DE STR (PerSeptive Biosystems, Framingham, MA). Aliquots of 3mL were mixed with 6mL of a saturated a–ciane-4-hydroxicianamic acid solution. The samples were crystallized at room temperature and analyzed under the following conditions: acceleration velocity 20 kV, laser intensity 1850J/cm², and 100 ns delay.

RESULTS AND DISCUSSION

Table 1 shows that all extracts from L. laevigatus tissues had no considerable toxicity (less than 1.7MU/g). On the other hand, extracts from S. spengleri showed high toxicity, ranging from 19.4 ± 0.68 up to 946.4 ± 60.9MU/g, always exceeding 10MU, the level at which flesh consumption is banned by sanitation authorities in other countries. All extracts from both species blocked the impulses evoked on the crustacean nerve fibers, and this effect reverted on washing in the same manner as standard TTX (data not shown), recorded, and analyzed by a computer program (5). Up until now, there are no data about behavior, biology, and seasonal studies of these two species, features that should explain the difficulty in collecting the specimens in some months through the year.

Table 1.
Mouse unit (MU) determination of extracts from muscle and skin plus viscera from L. laevigatus and S. spengleri collected in São Sebastião Channel between January 1996 and May 1997.

The presence of TTX, TDA, 4-epiTTX, and anhydroTTX in the tissues of L. laevigatus and S. spengleri was confirmed by HPLC (Figure 2) and mass spectrometry (Figure 3); these data were very similar to those obtained by other authors using different species of puffers and crabs (7,20,23,26-28).

Figure 2
. Chromatograms of semi-purified extracts of muscle (A) and skin plus viscera (B) from L. laevigatus, and muscle (C) and skin plus viscera (D) from S. spengleri. The peaks were obtained through a Shimpack CLC-ODS (0.6 x 250 mm) column with a linear gradient of 0.06N heptafluorobutiric acid and 0.001N ammonium acetate (pH 5.0) under 0.5mL/min flux, after derivatization with 4N NaOH. Tetrodotoxin (TTX), tetrodonic acid (TDA), 4-epi-tetrodotoxin (4-epi-TTX), and anhydrotetrodotoxin (anhydro-TTX).

Figure 3.
Representative mass chromatograms (m + H⁺) of peaks obtained from HPLC on the LC/MS MALDI-TOF Voyager-DE-STR (PerSeptive Biosystems, Framingham, MA). Aliquots of 3mL were mixed with 6mL of a saturated a–ciane-4-hydroxicianamic acid solution. The crystallized samples were analyzed under an acceleration velocity of 20kV, laser intensity 1850J/cm², and 100ns delay. The ion intensity of the base peak (100% relative intensity) was indicated in each mass chromatogram. The presence of (A) 320.03 Da - TTX; (B) 320.01 Da - 4-epiTTX; and (C) 302.10 Da - anhydroTTX was confirmed for L. laevigatus and S. spengleri tissue extracts

In Japan, because of rigorous studies of toxicity and biology of puffers, and increased attention to puffer fish selection and preparation, fatal cases of intoxication have been diminishing (8). Fishermen in the São Sebastião area consider L. laevigatus a non-toxic puffer; this was confirmed by our results. Tissues extracts from this species showed no considerable toxicity in mice, but traces of toxins are present at levels detected only by nerve bioassays. Frequent monitoring studies is a necessity in this area and in other Brazilian coastal states in order to allow the flesh of L. laevigatus to be commercialized and consumed with no risk to humans.

Based on symptoms in mice, TTX was the main toxic compound in both species, it also showed the greatest difference in toxin concentration (Table 1).

Sphoeroides spengleri showed an individual variation in toxicity. Skin and viscera were the most toxic tissues in all sampled months. The collecting sites also influenced the toxicity level of S. spengleri tissues. We found the most toxic species along the rocky shore of Portinho (Figure 4). In relation to public health, special attention should be paid to the fact that TTX content in muscle and skin plus viscera of S. spengleri always exceeded 10MU/g (Table 1). According to the Kawabata technique (12), flesh or other products from puffer fish can be eaten safely when their toxicity is less than 10MU/g. On the other hand, a person can easily eat more than 50g of flesh, therefore, the use of this species for food is dangerous based on the fact that ingestion of 10,000 MU (2mg) of TTX is lethal to an adult (25). Atlantic species are known to be less toxic than Pacific species, and based on our data, we suggest that the small S. spengleri is one of the most toxic species found in Atlantic waters.

Figure 4.
Geographic variation of toxicity of muscle (M) and skin plus viscera (S+V) extracts from Sphoeroides spengleri collected at two different rocky shores in the São Sebastião Channel. The statistical comparisons were made between toxicity of the same tissue extracts from the Cabelo Gordo and Portinho rocky shores. Data are plotted as the mean of 10-13 fish; vertical bars = standard error of the mean; *p<0.05.

As reported by Larson et al. (15), L. laevigatus is also a non-toxic species along the Atlantic coast of the United States. However, puffer fish toxicity is known to be is based on the feeding behavior and geographic distribution (17). Even inside the genus Lagocephalus, some species such as L. lunaris may have considerable toxicity (30). This is why we cannot state that all L. laevigatus from the entire Brazilian coastline are edible. In addition to the studies on L. laevigatus from São Sebastião Channel, further studies are necessary to know whether all specimens of this species have actually no toxicity in order to evaluate its use as food, and the possibility of future commercial exploitation. Associated with this, data on Fugu pardalis, F. stictonotus, F. niphobles, and other puffers showed wide regional variations in toxicity and high TTX levels, even in muscle (9-11,13,14,17). The muscles of specimens collected near Korea were more toxic than specimens from the Yellow Sea or Tachibana Bay, Japan. Our results indicate that regional variations in muscle toxicity of edible species of puffer fish must be more extensively studied. It is very important that Brazilian food sanitation authorities know which species can be considered edible and which cannot.

In this study, we confirmed the presence of TTX, 4-epiTTX, anhydroTTX, and TDA in extracts of muscle and skin plus viscera from both Brazilian puffers L. laevigatus and S. spengleri, as previously suggested by Oliveira (21). Although it is known that the source of TTX production and their analogs in marine organisms are symbiotic bacteria, the mechanisms involved in the incorporation, transport, and accumulation of these toxins in the target organs remain unknown.

ACKNOWLEDGEMENTS

We gratefully acknowledge the staff of the Dept. of Physiology, Institute of Biosciences and from Marine Biology Center, University of São Paulo, for their help and technical assistance. Thanks are also due to Prof. Dr. José Eduardo P. W. Bicudo, University of São Paulo, for his suggestions and reading of the manuscript. This research was supported in part by the Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

Received March 14, 2002

Accepted June 28, 2002

• 1 ALMEIDA VG., ROCHA CM. Registros de acidentes com peixes peçonhentos e/ou venenosos. Rev. Soc. Bras. Toxicol, 1989, 2, 49-51.
• 2 BURKLEW MA., MORTON RA. The toxicity of the Florida Gulf puffers, genus Sphoeroides Toxicon, 1971, 9, 205-10.
• 3 CESTÉLE S., CATTERALL WA. Molecular mechanisms of neurotoxin action on voltage-gated sodium channels. Biochimie, 2000, 82, 883-92.
• 4 CORRÊA MF., SILVA LR., KONOLSAISEN JF., ASSANUMA MH., ABILHOA V. Determinação da toxicidade oral do baiacu-pintado Sphoeroides testudineus (Osteichthyes, Tetraodontiformes) em camundongos e sintomatologia da intoxicação em camundongos (Mus musculus) e cobaias (Cavia porcelus). Arq. Biol. Tecnol. (Curitiba), 1990, 33, 413-38.
• 5 DEMPSTER J. Computer analysis of electrophysiological signals Strathclyde: Academic Press - Harcourt Brace Jovanovich Publishers, 1993. 228p.
• 6 DO HK., Kogure K., Simidu U. Identification of deep sea sediment bacteria which produce tetrodotoxin. Appl. Environ. Microbiol., 1990, 56, 1162-3.
• 7 FREITAS JC., SATO S., OGATA T., KODAMA M. Guanidine neurotoxins are released with the digestive fluid of crabs (Crustacea, Brachyura). Toxicon, 1995, 33, 201-8.
• 8 HASHIMOTO Y. Marine toxins and other bioactive marine metabolites Tokyo: Jpn. Sci. Soc. Press, 1979. 247p.
• 9 HWANG DF., CHENG CA., TSAI YH., JENG SS. Tetrodotoxin associated food poisoning due to unknown fish in Taiwan between 1988-1994. J. Nat. Toxins, 1995, 4, 165-71.
• 10 KANOH S., NOGUCHI T., KAMIMURA S., HASHIMOTO K. A survey of toxicity of the pufferfish, Fugu pardalis, inhabiting the Sanriku Coast. J. Food Hyg. Soc. Jpn., 1984, 25, 24-9.
• 11 KANOH S., NOGUCHI T., MARUYAMA J., KAMIMURA S., HASHIMOTO K. Toxicity of the pufferfish Fugu stictonotus collected from the Sanriku coast. Nippon Suisan Gakkaishi, 1985, 51, 121-5.
• 12 KAWABATA T. Puffer toxin. In: THE JAPANESE FOOD HYGIENE ASSOCIATION. The manual for the methods of food sanitation tests II Tokyo: THE JAPANESE FOOD HYGIENE ASSOCIATION., 1978: v.2, 232-40.
• 13 KODAMA M., OGATA T. Toxicity of a fresh water puffer Tetrodon leiurus Bull. Jpn. Soc. Sci. Fish, 1984, 50, 1949-51.
• 14 KODAMA M., OGATA T., KAWAMUKAY K., OSHIMA Y., YASUMOTO T. Toxicity of muscle and other organs of five species of puffer collected from the Pacific coast of Tohoku area of Japan. Bull. Jpn. Soc. Sci. fish, 1984, 50, 703-6.
• 15 LARSON ER., LALONE RC., RIVAS LR. Comparative toxicity of the Atlantic puffer fishes of the genera Sphoeroides, Lactophrys, Lagocephalus and Chilomycterus Fed. Proc. Fed. Am. Socs. Exp. Biol, 1960, 19, 388.
• 16 LARSON E., LALONE RC., DE VILLEZ EJ., SIMAN JR. R. Animal Toxins New York:: Pergamon Press, 1967. 428p.
• 17 MATSUI T., SATO H., HAMADA S., SHIMIZU C. Local variation of toxicity of the puffer fish Fugu niphobles. Bull. Jpn. Soc. Sci. Fish, 1982, 48, 1179.
• 18 MATSUMURA K. Reexamination of tetrodotoxin production by bacteria. Appl. Environ. Microbiol., 1995, 61, 3468-70.
• 19 MOSHER HS. The chemistry of tetrodotoxin. In: BY KAO CY., LEVINSON SR. Eds. Tetrodotoxin, saxitoxin and the molecular biology of the sodium channel New York:: Annals of the New York Academy of Sciences, 1986: v.479, 32-43.
• 20 NOGUCHI T., ALI AE., ARAKAWA O., MIYAZAWA K., KANOH S., SHIDA Y., NISHIO S., HASHIMOTO K. Toxicon, 1991, 29, 845-55.
• 21 OLIVEIRA JS. Determinação dos níveis de tetrodotoxina em peixes marinhos da família Tetraodontidae (Osteichthyes, Teleostei) São Paulo: Universidade São Paulo, Instituto de Biociências, 1998. 143p. [Tese-Doutorado].
• 22 SATO S., KOMARU K., OGATA T. Occurrence of tetrodotoxin in cultured puffer. Nippon Suisan Gakkaishi, 1990, 56, 1129-31.
• 23 SHOJI Y., YOTSU YAMASHITA M., MIYAZAWA T., YASUMOTO T. Electrospray ionization mass spectrometry of tetrodotoxin and its analogs: liquid chromatography/mass spectrometry, tandem mass spectrometry, and liquid chromatography/tandem mass spectrometry. Anal. Biochem., 2001, 290, 10-7.
• 24 SIMIDU U., NOGUCHI T., HWANG DF., SHIDA Y., HASHIMOTO K. Marine bacteria which produce tetrodotoxin. Appl. Environ. Microbiol., 1987, 56, 1162-3.
• 25 TANI I. A study of the toxicity of Japanese fugu Tokyo. 1945. 103p.
• 26 YASUMOTO T., MICHISHITA T. Fluorometric determination of tetrodotoxin by high performance liquid chromatography. Agric. Biol. Chem., 1985, 49, 3077-80.
• 27 YASUMOTO T., NAKAMURA M., OSHIMA Y., TAKAHATA J. Construction of a continuous tetrodotoxin analyzer. Bull. Jpn. Soc. Sci. Fish, 1982, 48, 1481-3.
• 28 YASUMOTO T., NAGAI H., YASUMURA D., MICHISHITA T., ENDO A., YOTSU M., KOTAKI Y. Interspecies distribution and possible origin of tetrodotoxin. In: KAO CY., LEVINSON SR. Eds. Tetrodotoxin, saxitoxin and the molecular biology of the sodium channel New York: Annals of the New York Academy of Sciences, 1986: v. 479, 44-51.
• 29 YASUMOTO T., ENDO A., YASUMURA D., NAGAI H., MURATA M., YOTSU M. Bacterial production of tetrodotoxin and its derivatives. In: GOPALAKRISHNAKONE P., TAN CK. Eds. Marine toxins: progress in venom and toxin research Singapore: National University of Singapore. IST Asia-Pacific Section, 1987: 3103.
• 30 YIP LL., CHIU KW. Toxicity of the common puffer fish in Hong Kong. Experientia, 1981, 27, 668-9.
• 31 Yotsu M., ENDO A., YASUMOTO T. An improved tetrodotoxin analyzer. Agric. Biol. Chem, 1989, 53, 893-5.
• 32 YOTSU M., YAMAZAKI T., MEGURO Y., ENDO A., MURATA M., NAOKI H., YASUMOTO T. Production of tetrodotoxin and its derivatives by Pseudomonas sp., isolated from skin of a pufferfish. Toxicon, 1987, 25, 225-8.

Publication Dates

• Publication in this collection
  09 Dec 2003
• Date of issue
  2003

History

• Received
  14 Mar 2002
• Accepted
  28 June 2002