Toxicity of the Red Sea pufferfish Pleuranacanthus sceleratus “El-Karad”

ARTICLE IN PRESS

Ecotoxicology and Environmental Safety 56 (2003) 367–372

Toxicity of the Red Sea pufferfish Pleuranacanthus sceleratus ‘‘El-Karad’’ M. El-Sayed,a G.A. Yacout,a, M. El-Samra,b A. Ali,c and S.M. Kotbb a

Biochemistry Department, Faculty of Science, Alexandria University, Alexandria, Egypt b National Institute of Oceanogrophy and Fisheries, Alexandria, Egypt c Department of Marine Science, Faculty of Science, Suez Canal University, Egypt

Received 15 May 2002; received in revised form 9 October 2002; accepted 28 October 2002

Abstract The pufferfish Pleuranacanthus sceleratus (El-Karad) represents serious public health problems, because of its responsibility for many incidents in Egypt, especially in the Suez Gulf. In the present study, samples of this fish were collected monthly and the toxins were extracted from gonads, liver, digestive tract, muscles, and skin, then purified and identified using TLC and electrophoresis. The alkaline hydrolyzates of these toxins were also detected using UV absorption and GC-mass spectra of their trimethylsilyl derivatives. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Pufferfish; Toxins; Tetrodotoxin

1. Introduction It has been noted that when humans encounter marine creatures a variety of maladies may occur, ranging from dermatitis to life-threatening trauma, allergy, or intoxication with poisonous animals (Brown and Shepherd, 1992). Moreover, animal venoms and poisons have been investigated with respect to their biological relevance (Harbermehl and Krebs, 1986). These substances have been revealed to be fungicidal, growth-inhibitory, antiviral, antibiotic, antitumor, analegesic, and cardio-inhibitory (Halstead et al., 1988). Previous data revealed that pufferfish toxin, tetrodotoxin (TTX), a potent neurotoxin, is widely used in many laboratories as an important pharmacological reagent because of its ability to block selectively the sodium channels on the nerve membrane (Moore et al., 1966; Narahashi et al., 1994). This toxin has been isolated from various animals (Noguchi et al., 1985; Ali et al., 1990). Pufferfish poisoning is a widespread phenomenon, not confined to Egypt, and involves different species of pufferfish in different parts of the world (Tanner et al., 1996; Hwang et al., 1995). In some places it is assumed that the toxicity is only in gonads and liver, and that proper preparation will eliminate 

Corresponding author. E-mail address: galila [email protected] (G.A. Yacout).

toxicity (Elam et al., 1977). The present work reports that this is not true for the pufferfish of Egypt. Despite the rare use of pufferfish as a food in Egypt, there is a record of poisoning cases in Suez City (Ali, 1996). This study was undertaken to evaluate the toxicity of five organs: gonads, liver, muscles, digestive tract, and skin. In addition, the extracted toxins were partially purified and identified using TLC, electrophoresis, UV spectrophotometr, and GC-mass spectroscopy. This work covers Suez City, in the northwestern part of the Red Sea.

2. Materials and methods 2.1. Sampling A total of 45 specimens of pufferfish, Pleuranacanthus sceleratus, were collected monthly from September 1990 through May 1991. Directly after collection, the fresh samples were weighed and immediately frozen at 201C. 2.2. Animal used Male Swiss albino strain mice with a body weight of 2072 g were obtained from National Research Center, Dokki, Cairo, Egypt.

0147-6513/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0147-6513(02)00142-2

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2.3. Test for toxicity

skin) was determined according to the standard mouse bioassay method of Kawabata (1978). One gram from each organ was homogenized with 4 mL of 0.1% acetic acid, boiled on a water bath for 10 min, cooled, and

The toxicity of different organs obtained from individual fish (gonads, liver, muscles, digestive tract,

Table 1 Fish size and sex and relative toxicity of each organ (MU/g) by season for P. sceleratus Date of collection

Sept. 1990

Total length (cm)

Body weight (g)

Sex

Toxicity (MU/g) Muscle

Skin

Liver

35.5 44 49.5 65

463 972 1234 2855

M F F F

7 9 9 6

11 9 10 4

18 9 11

Oct. 1990

50 45 50 53 46

3600 2700 3620 4020 2730

M M F F F

68 30 64 17 17

Nov. 1990

59 47 34.5 51 25

4250 2725 1500 3760 177

M F F F F

40 40 38 39 35 59 56 60

500 600 455 500 381 2250 1500 2000

M M M M M F F F



12 50

Jan. 1991

49 51.5 53 63 59 55

1284 1513 1662 3075 2142 2120

M F F F F F

13 9 41 6 13 9

Feb. 1991

38.5 39.5 40.5 40.5 37.5 40 45

644 676 716 737 628 682 957

M M M M F F F

Dec. 1990

Gonads

Digestive tract

104

20





48 12

9



56 51 57 13 13

223 38 192 48 28

237 50 158 90 338

144 83 161 31 28

7

7

28



25 4

25

















6 41

9 119

20 61

125 752

20 73

3

7 5



12

5































4

6

7



9









19 14

18 49



119

18 31

16 16 40 12 7 12

6 94 21 10 98 172

11 115 30 129 271 465

32 48 13 12 108 177

5



4

11

6























14















5

















7



Mar. 1991

45 44 47 46

882 982 1179 1052

M M F F

127 11 27 12

35 4 13 9

246 10 38 49

121 386 160 236

221 11 15 89

Apr. 1991

42 45 53

688 943 1931

M M F

10

3 4 4

11



11



23 21

5

39 58 64

772 2289 3350

M F F

26 9

9 15 5

3 98

4 153 70

4 75 9

May. 1991



Not detected.

 



5





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centrifuged off, and then 1 mL of the supernatant obtained was injected intraperitoneally into a group of three male albino mice. The median death time (period between injection and death) was used to calculate the number of mouse units (MU) (Table 1). One mouse unit is defined as the amount of toxin that kills a 20 g male mouse by 30 min after intraperitoneal injection. 2.4. Isolation and partial purification of toxin Organs pooled from all specimens were collected (478 g gonads, 2000 g liver, 1015 g digestive tract, 15,500 g muscles, 3250 g skin) and homogenized separately with 1 vol. of 1% acetic acid. Each homogenate was centrifuged off at 3000 rpm for 20 min (Ali et al., 1990). The pellet obtained was collected and extracted twice with the same volume of 1% acetic acid. Each combined extract was concentrated separately under reduced pressure and defatted with CH2Cl2. The aqueous obtained layer was concentrated, adjusted to pH 4–5 with 1 N NaOH solution, and then treated with activated charcoal. The adsorbed toxin was eluted with 3 vol. of 1% acetic acid in 20% ethanol. The eluates were combined and evaporated under vacuum. Further purification was carried out according to Kotaki et al. (1981), by gel filtration using a column (2.4  120 cm2) packed with Bio-Gel P-2 (Bio-Rad Laboratories, Richmond, CA, USA). Elution of the toxin was carried out with 0.03 N acetic acid at a flow rate of 60 mL/h; then the toxicity of collected fractions were determined by the mouse bioassay. 2.5. Thin-layer chromatography of toxins TLC was performed on silica gel 60-F254 precoated plates (Merck) with the butanol:acetic acid:water (2:1:1 v/v) solvent system. After development, the plates were sprayed with 10% KOH, then heated at 1101C for 10 min, according to Onoue et al. (1984). The developed toxins were visualized as yellow spots under UV light at 365 nm. Authentic standards, TTX and tetrodonic acid (TDA) (Fig. 1), were kindly supplied by Dr. A.E. Ali,

369

Department of Marine Science, Faculty of Science, Suez Canal University. 2.6. Electrophoresis Electrophoresis was performed on 76  60-mm2 cellulose acetate strips (Helena) in 0.08 M Tris–HCl buffer, pH 8.7, under a constant current of 0.8 mA/cm for 10 min, Toxins were detected in the same manner as in TLC (Saito et al., 1985). 2.7. Alkaline hydrolysis of toxins About 500 mL of 1% acetic acid containing the toxins from each organ was hydrolyzed by heating with 2 mL of 2 N NaOH for 45 min, according to the method of Suenaga and Kotoku (1980). Standard TTX was hydrolyzed in the same manner. 2.8. Detection of the produced C9 base The hydrolyzates obtained were studied separately by UV spectrophotometry (Beckman Du-6), according to Narita et al. (1987). Also, the C9 base produced was converted to its trimethylsilyl (TMS) derivative by dissolving it in 0.5 mL of a mixture of bistrimethylsilyl acetamide, trimethylchlorosilane, and pyridine (2:1:1), and then analyzing it in a GC-mass spectrometer (SSQ 7000) with fused silica capillary column (30  0.2 mm2) of 3% DB-1, the column temperature was programmed from 1601C to 2501C at the rate of 51C/min, and the flow rate of inlet helium was maintained at 40 cm3/s, according to Narita et al. (1981) and Noguchi et al. (1981).

3. Results Forty-five P. sceleratus specimens consisting of 19 males and 26 females were studied to investigate the toxicity of different organs. The toxicity results are given in Table 1. Noted that the toxicity of the gonads was the highest when compared with digestive tract, liver,

Fig. 1. TTX and TDA.

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muscles, and skin, giving 752, 221, 246, 127, and 119 MU/g, respectively. The results summarized in Table 1 revealed a seasonal variation in toxicity particularly with respect to gonads. Ovaries contained 752 MU/g (the highest toxicity) in November 1990. Whereas testes contained 386 MU/g in March 1991. However, the values were 465 MU/g in January 1991 and 237 MU/g in October 1990 for ovaries

Table 2 Steps of partial purification of toxin separated from different organs of P. sceleratus Step

Total toxicity (MU)/specify toxicity (MU/g) Gonads

Digestive tract

Liver

Muscles

Skin

Defatting

43,498 91

13,175 13

10,060 50

542,500 35

94,250 29

Charcoal

1232 123

314 29

1852 69

797 44

4501 90

Bio-Gel

573 76

544 74

6080 357

1377 110

2292 43

TDA

and testes, respectively. Thus, for P. sceleratus, ovaries were the most toxic, followed by testes. In addition, the toxin isolated from each organ was defatted with CH2Cl2 and purified using activated charcoal followed by gel filtration using Bio-Gel P-2 column chromatography. The total and specific toxicities of toxin separated from each organ were calculated as shown in Table 2. It was found that after gel filtration, there was some loss in toxicity in the case of gonads and skin obtained during processing, but in the case of digestive tract, liver, and muscles, specific toxicity increased. The isolated toxins were characterized by TLC (Fig. 2), and showed the same pattern: a single spot with an Rf value of 6.4 cm corresponding to that of authentic TTX. In the case of muscles, liver, and digestive tract, another spot with an Rf value of 4.2 cm appeared, coinciding with that of TDA. As seen in Fig. 3 the electrophoretic patterns of the purified toxin isolated from the five organs were detected as one clear fluorescent yellow spot with a migration distance of 5.7 cm, when visualized with 10% KOH, suggesting that these spots were associated with TTX. Another spot, occurring only for toxin from muscles, liver, and digestive tract, had a migration distance of 3.1 cm, which agreed well with that of authentic TDA.

TTX TDA

TTX

G

M G

DT

M

S

DT

L

S

L

Rf 0

9

Fig. 2. TLC of the extracted toxins from different organs of P. sceleratus. Along with authentic TTX and TDA. G, gonads; M, muscles; DT, digestive tract; S, skin; L, liver. 1-BuOH:AcOH:H2O (2:1:1).

0

7 Distance of migration (cm.)

Fig. 3. Electrophoresis of the extracted toxins from different organs of P. sceleratus along with authentic TTX and TDA.

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100

50

14:10

14:35

15:00

15:25

0 350

370

390

410

370

390

410

100

50

14:10

14:35

15:00

15:25

0 350

Retention time (min) Fig. 4. GC-mass spectra of the TMS derivative of the alkaline hydrolyzate of gonad toxin (top) and the corresponding C9 base from TTX (bottom).

In UV spectrophotometry, the alkaline degradation product of toxin isolated from the five organs exhibited an absorption maximum at 272–274 nm, which agreed with that of the C9-base derived from authentic TTX. Gas chromatographs of the TMS derivatives of the alkaline hydrolyzate of gonadal toxin and the derivatives of authentic TTX are in Fig. 4. The same pattern was obtained for the alkaline degraded product of toxin isolated from liver, muscles, skin, and digestive tract. Peaks appeared at the same retention time of 14.48 min, which was similar to that of C9 base. The released peaks when subjected to mass spectrometry revealed essentially the same mass spectra, which featured fragment ions at m=z 407(M+), 392(100%), and 376.

4. Discussion The results of the present study indicate that all the organs are toxic but to varying degrees indicating an unequal distribution of the toxin. Also, the toxicity of an organ varies with time of collection and sex in agreement with the previous data of Ali et al. (1995). Some individuals exhibited no toxicity (non-significant values) in some months, which could be attributed to the physiological condition of the individual, or to the different vital activities of the fish such as fasting, or to different diets in different periods. Also, the level of toxin varied among the individual puffers and during different seasons, especially in the ovary which had the highest toxicity scores. This may be

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due to the different diets of the fish in different periods. The result obtained coincides well with that obtained by Kao (1966), Fuchi et al. (1991), and Ali et al. (1995), who studied different organs of other pufferfish and showed that puffer toxins were concentrated in ovaries and liver, with lesser amounts present in the digestive tract and skin. This suggests that this pufferfish may use TTX as a defense agent for offspring (Khora et al., 1990). In addition, the results obtained indicate that muscle, the edible part of fish, contained 127 MU/g in March, so about 24 g of muscle is enough to kill a human being, since previous data obtained by Halstead (1965) indicated that can 3000 MU is fatal to humans. This reflects the danger of using this edible part of fish as a food. On the other hand, after isolation and purification of the isolated toxins, which is carried out by defatting with CH2Cl2, activated charcoal treatment, and Bio-Gel P-2 column chromatography, some loss in toxicity occurred in the case of gonads and skin. Meanwhile, there were increases in toxicity in digestive tract, liver and muscles. This increase is probably due to the labile toxin TDA, which is easily converted to TTX. The TLC and electrophoretic behavior of the extracted toxins clearly indicates the presence of TTX in all organs. In addition, muscle, skin, and digestive tract showed TDA. These results coincide well with those obtained by Ali (1996) on toxin extracted from the liver of the pufferfish Arothron hispidus. UV and GC-mass spectra of the alkaline degradation product of the extracted toxin, suggest that these toxins possess a quinazoline skeleton similar to that of the C9 base derived from authentic TTX, in agreement with Saito et al.(1985) and Ali (1996), who studied the toxins released from the pufferfish Fugu niphobles and A. hispidus, respectively. These observations on the toxicity of this Egyptian pufferfish, revealing toxicity in all organs, especially, muscle may be expanded to other species in other parts of the world, and clearly indicate the danger of using this fish as food.

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