äi41-0101(94)00113-8
Toxfcar, vol . 3x, No. Iz pp . 1573-1579,1994 (D 1994 Elsevier science lAd printed in Great Britain . A11 rim reserved 0041-0101194 S7.00+ .00
GONYAUTOXIN-3 AS A MINOR TOXIN IN THE GASTROPOD NIOTHA CLATHRATA IN TAIWAN DENG FWu HWANG, CHAD AN CHENG and SEN SHYoNG JENG
Department of Marine Food Science, National Taiwan Ocean University, Keelung, Taiwan, Republic of China
(Receüvd 19 May 1994; accepted 22 July 1994)
D. F. HWANG, C. A. CHENG and S. S. JENG. Gonyautoxin-3 as a minor toxin in the gastropod Niotha clathrata in Taiwan. Toxicon 32, 1573-1579, 1994.-Paralytic toxicity was detected in the gastropod mollusc Niotha clathrata collected from South Taiwan in April and November 1993. Each seasonal toxin was partially purified from toxic specimens of N. clathrata by ultrafiltration using a membrane (Diaflo YM-2), followed by chromatography on a column (Bio-Gel P-2). Two toxin fractions (I and II) were then obtained for each seasonal shell toxin . The ratio of fraction I to fraction II for each seasonal shell toxin was about 4 :1 according to tetrodotoxin bioassay. Based on analyses by TLC, electrophoresis, and HPLC, fraction I toxin contained tetrodotoxin and its derivative anhydrotetrodotoxin, and fraction II toxin contained gonyautoxin-3 for each seasonal shell toxin. AMONG marine biotoxins, tetrodotoxin (TTX) and paralytic shellfish poison (PSP) are well known as sodium-blocking neurotoxins . TTC and/or TTX-related compounds have been reported to occur in gastropod molluscs including the trumpet shell Charonia sauliae (NARITA et al., 1981, 1989), frog shell Tutufa lissostoma (NoGucm et al., 1984), Japanese ivory shell Babylonia Japonica (NoGucm et al., 1981 ; YAsumoTo et al., 1981), Zeuxis siquijorensis (NARITA et al., 1984), and Niotha clathrata (JEoN et al., 1984) . The gastropod molluscs Naticidae, such as Natica lineata, N. vitellus and Poilnices didyma, and gastropods Rapana rapiformis and R. venosa venosa collected from Taiwan also contain TT{ (HWANG et al., 1990, 1991a-c) . Moreover, the gastropod Niotha clathrata and Zeuxis scalaris collected from Taiwan was found also to contain TTX and an unknown toxin (HWANG et al., 1992a-c). This unknown toxin obtained from N. clathrata was found to be similar to TTt according to physicochemical characteristics, but its alkaline degradation product was distinct from that of TTX (HWANG et al., 1992a) . These properties prompted us to compare this unknown toxin with PSP components which are identified as major toxins in the purple clam Soletellina diphos (HWANG et al., 1987). Therefore, this work was undertaken to compare the unknown toxin with authentic PSP components, and to evaluate the seasonal variation of toxin composition in the shell N. clathrata . Materials
MATERIALS AND METHODS
In total, 165 and 200 specimens of Nlotha clathrata were collected from Tungkang, Pingtung Prefecture, South Taiwan in April and November 1993, respectively . The respective average body lengths and weights were 1573
1574
D.
F. HWANG et al.
2.46 t 0.03 (mean f S.D .) cm and 3.50 t 0.12 g in April, and 2.36 t 0.05 cm and 2.98 t 0.18 g in November. The specimens were kept frozen at -20°C until used for toxicity assay. Assay method for toxicity
The edible part of all specimens was homogenized and examined in triplicate for toxicity according to a modified mouse assay method for TTX (HWANG and JENG, 1991). The dose-death time curve of N. clathrata toxin was established with the ICR (Institute of Cancer Research) strain, and also with gonyautoxin-1-4 (GTX-1-4) obtained from the purple clam Soletellina diphos (HWANG et al., 1987). Lethal potency is expressed in mouse units: one mouse unit (MU) is defined as the amount of toxin required to kill an ICR strain male mouse (20 g) in 30 min after i.p . injection. Statistical analyses were performed by the matched-pair t -test. Isolation of toxin
Specimens were combined, homogenized for 5min with about three volumes of acetic acid (1 %) in methanol, and centrifuged (3000 x g, 20 min). This operation was repeated twice more . The supernatants were combined, concentrated under reduced pressure at 40°C, and defatted with dichloromethane. The aqueous layer was concentrated and filtered through a membrane (Dafflo YM-2, AmioDn), having a limit at 1000 mol. wt . The filtrate was applied to a column (Bic-Gel, 2 x 94 cm) which was developed with 0.03 M acetic acid. Toxic fractions were combined, freeze-dried,dissolved in a little water, and submitted to subsequent analyses . Authentic TTX and anhydrotetrodotoxin (anh-TTX) (Gom et al., 1965; HWANG et al., 1988) obtained from the liver of the puffer Feegu oblongus were used as reference standards. Authentic GTX-1-4, saxitoxin (STX) and neoSTX obtained from the purple clam Soletellina diplws and the crab Zosinnrs aeneus (DAtoo et al., 1985; HWANG et al., 1987) were also used as reference standards. 71än-layer chromatography
Thin-layer chromatography (TLC) was performed on 5 x 20 cm (thickness 2 mm) silica gel 60 F~,-precoated plates (Merck) with two solvent systems: pyridine-ethyl acetate-acetic acid-water (15 :5 :3 :6) and 1butanol~cetic acid-water (2 :1 :1). Toxins were visualized as blue, green or yellow fluorescent spots under a UV lamp (365 nm) after spraying the plate with 10% KOH or 1% H2O2, and then heating at 110°C for 10 min. Electrophoresis
Electrophoresis was performed in 5 x 18 cm cellulose acetate strips (Chemetron) in Tris-HCI buffer (0 .08 M, pH 8.7) under a constant current of 0.8 mA/cm for 1 hr. Toxins were visualized as described for TLC. High-performance liquid chromatography
High-performance liquid chromatography (HPLC) was performed on a reversed-phase column (YMC-Pack AM-314 ODS, 6 mm I.D . x 300 mm) (NAGAsHimA et al., 1987). The mobile phase was sodium 1-heptane sulfonate (2 mM) in methanol (1%)-potassium phosphate buffer (0 .05 M, pH 7.0) for TTX and GTX assays, and in methanol (20%)-potassium phosphate buffer (0 .05 M, pH 7.0) for STX assay. The flow rates were 1 ml/min for TTX assay and 0.8 ml/min for GTX and STX assays. The eluate was mixed with an equal volume of NaOH (3 N) and heated in a reaction coil (Teflon tube, 3 mm I.D . x 10 m) at 99°C, and the intensity of fluorescence was measured at 505 run with 380 mm excitation for TTX assay. For GTX and STX assays, the eluate was mixed with an equal volume of periodate reagent. The fluorogenic reaction was performed at 65°C for 0.7 min. The intensity of fluorescence was measured at 388 nm with 344 excitation . RESULTS
The elution profile of the toxin on a column (Bie-Gel P-2) chromatograph is shown in Fig. 1. Two toxic fractions (I and II) were obtained from each seasonal shell toxin of N. clathrata. The toxicity of shell specimens was 9.21 f 0.33 (mean t S.D .) MU and 9.70 f 0.30 MU in April and November, respectively, and showed no seasonal variation. The respective total toxicities of fractions I and II were 900 t 10 MU (400 f 5 MU/mg) and 200 f 6 MU (280 f 8 MU/mg) in April, and 1100 f 20 MU (380 t 6 MU/mg) and 300 t 7 MU (290 t 5 MU/mg) in November in N. clathrata toxin (Table 1). The respective percentages of fractions I and II were 81 .8 f 1.0% and 18.2 f 0.6% in April, and 78.6 t 1 .4% and 21.4 f 0.5% in November in the shell toxin. It was found that fraction II toxin significantly increased in November (P < 0.05).
GTX-3
as Minor Toxin in Niotha clathrata
1575
A) November SO .-
=N
60
Fraction I
E
v
40
U 12
20
06 80
90
100
110
120
B) April 80
60 É
Fraction I
40 U ~X O F
Fraction No . (5 ml/Fr.) FIG. 1 .
Fa.unoN PROFILE of Toxin FROM Niotha clathrata couEc= IN NOvEMBER (A) AND (B) 1993 ON A COLUMN (BIO-GEL P- Z) USING ACETIC ACID (0 .03 M) As ELuENT .
APRIL
As shown in Fig. 2, electrophoresis of fraction I from each seasonal shell toxin showed two spots, which were indistinguishable from TTX and anh-TTX, both in migration distance (8.0 and 5.5 cm) and in fluorescent colour (yellow) . Fraction II from April shell toxin showed three spots, and two spots were indistinguishable from GTX-3 and GTX-2, both in migration distance and in fluorescent colour, whereas fraction II from the November shell toxin showed one spot, which coincided with GTX-3. However, the spot which showed the retention distance of 2.1 em and blue fluorescent colour in fraction II from the April shell toxin was unidentified . In TLC, fraction I from each seasonal shell toxin exhibited one spot, which coincided with TTX and/or anh-TTX, according to two solvent systems . Fraction II from the April and November shell toxins showed two and one spots in TLC, respectively . Among them,
1576
D. F. HWANG et
al.
TABLE 1 . PURIFICAT)ON of TOXIN FROè1 Niotha clathrata COLLECTED AT TÜNGKANG, PINGTUNG PREFECTURE, IN APRIL AND NOVEMBER 1993
Total toxicity (MU)
Steps AcOH (I %~-MeOH (99%) extract Ultrafiltration Bio-Gel P-2 Faction 1
Apr. Nov . 1520 f 50 1940 t 60 (9.2l t 0.33)' (9 .70 t 0.30) 1440 f 41 1760 f 45 1100± 15 1400 f 24 900 t 10 1100 f 20 (81.8 f 1.0%)t (78.6 f 1 .4%) Fraction 11 200 f 6 300 f 7 (18.2 f 0.6%) (21.4 f 0.5%) *Mean f S.D . of toxicity per shell specimen . tThe percentage of each fraction toxin in total shell toxin.
Specific toxicity (MU/mg) Apr. Nov.
400 t 5
380 t 6
280 f 8
290 f 5
the major spot was identified as GTX-3, and the minor spot in the April shell toxin was identified as GTX-2 (data not shown) . In HPLC, fraction I of each seasonal shell toxin contained two signals which had the same retention periods (9 .8 and 12.2 min) as those of TTX and anh-TTX, respectively (Fig. 3). Fraction II of each seasonal shell toxin contained one or two signals that had the same retention periods (15.3 and 17.0 min) as those of GTX-3 and GTX-2, respectively (Fig. 4). Further, fraction 11 of each seasonal shell toxin contained neither STX or neoSTX (data not shown) . The dose-death time curve of fraction II toxin from N. clathrata collected on April 1993 in mice was found to agree with that of the purple clam toxin (authentic GTX) . Because purple clam toxin contained mainly GTX-3, along with trace GTX-2, GTX-1 and GTX-4, fraction II toxin from N. clathrata seemed to contain the same toxin compositions as those of purple clam toxin (data not shown). According to these data, we conclude that fraction I toxin from N. clathrata contained TTX and anh-TTX, and fraction II toxin from N. clathrata contained GTX-3 and GTX-2.
GTX4,1,a,2
I
e
neoSTX, STx1
anh-TTX, TTX I
I I I I
I toxin
~. [Fr. II toxin Nov..
I toxin
Fr. II toxin
0
MG .
2.
0:
0
A
0
0
08 D
0
0
0
o
4 2 6 8 Distance of migration (cm) blue ;
@ : green ;
0:
10
yellow ;
CELLULOSE ACETATE MEMBRANE ELECTROPHORE= of TOXIN FROM Nfotha clathrata COLLECTED Ev APR& AND NovEe®ER 1993, Wmr AUTHENTIC GTX, STX AND TTX.
The strip was sprayed with Hz02 (1%) or KOH (10%) and heated at 110°C for 10 min.
GTX-3 as Minor Toxin in Niotha clathrata November
'âo c ç 0 u c u0
April Fr. I toxin
0
TTX
anh-TTX
Retention time (min) Fto. 3 . HPLC oP FRAcnoN I To3aN Paom Niotha clathrata coLtEC= nv NovEMBne (urane) AND APan. (mwDLE) 1993, wrrH AuTtmNnc TTX (LowER).
c 0 c_ .o 0 c 0 X 0
Retention time (min)
Fto. 4. HPLC OF t+RACMON II ToxiN FROM Niotha clathrata couEcrt n tN NovsuaEx (uPPER) AND APan. (1UDDIE) 1993, wrrx AtrrEnrNTtc GTX (t .own<) .
1577
1578
D. F . HWANG et al. DISCUSSION
In this work, the toxicity of N. clathrata collected from Tungkang, Pingtung Prefecture in April and November 1993 showed no clear seasonal variation. However, the toxicity of N. clathrata collected during autumn and spring in 1989-1990 was greater than in other seasons (HWANG et al., 1992b). The environment and food chain of N. clathrata possibly altered during different periods of growth . Moreover, the amounts of fraction I and II toxins showed significant seasonal variation. The shell accumulates more fraction II toxin in winter . As previously reported (HWANG et al., 1992a-c), TTX was the major toxin, and an unknown toxin was a minor toxin in the gastropod N. clathrata. In this work, we found that TTX was also the major toxin, and GTX-3 was a minor toxin in this gastropod. Hence, we reasonably conclude that GTX-3 was the responsible toxin in fraction II toxin previously reported as unknown. Fraction II toxin also contained GTX-2 in the April shell specimen . According to these results, the gastropod N. clathrata contained mainly TTX, with minor GTX-3 and GTX-2. Until now, the occurrence of PSP in marine organisms has been invariably connected with the dinoflagellate Alexandrium spp. (OGATA et al., 1987; OsHwA et al., 1987 ; UEDA et al., 1982). Although the gastropod N. clathrata was known to contain TTX (HWANG et al., 1992x), PSP components were first identified as a minor toxin in this shell. Even in the same place, some gastropods such as Natica fneata, N. vitellus and Poilnices didyma were found to contain TTX (HWANG et al., 1990, 1991 a, b), and the purple clam S. diphos (HWANG et al., 1987) was found to contain PSP. Hence, shellfish accumulate TTX and/or PSP depending on the varied food chain in the marine environment. Acknowledgement-We thank the National Science Council, R.O .C . (Grant No. NSC 82-0409-8019-034) for a grant. REFERENCES DAtao, K ., Uzu, A., ARAKAwA, 0., Norman T ., SurA, H. and Hmmmoro, K . (1985) Isolation and some properties of neosaxitoxin from a xanthid crab Zosinuus seneus . Nippon Suds. Gakk. 31, 309-315. Goro, T., Kma, Y., TAKAHAsin, S. and HIRATA, Y. (1965) Tetrodotoxin . Tetrahedron 21, 2059-2088 . HWANG, D . F . and JENG, S. S . (1991) Bioassay of tetrodotoxin by using ICR strain male mouse. J. Chin. Biochem . Soc. 20, 80-86. HWANG, D . F., NoGucm, T., NAGAsimiA, Y., LiAo, I . C. and HAmmsoro, K. (1987) Occurrence of paralytic shellfish season in the purple clam Soletellina diphos (bivalve). Nippon Sais . Gakk. 53, 623-626. HwANo, D. F ., NooucFn, T., AAAKAwA, O ., ABE, T. and HmFmcoro, K . (1988) Toxicological studies on several species of puffer in Taiwan. Nippon Suis. Gakk. 54, 2001-2008. HwANo, D. F., CttuEH, C. H . and JENG, S . S . (1990) Occurrence of tetrodotoxin in the gastropod mollusk Natica lineata (lined moon shell) . Toxicon 28, 21-27 . HwANo, D. F ., CttuEH, H. C., TAi, K . P ., Luv, L . C . and JENo, S . S . (1991 a) Seasonal and regional variations of toxicity in the lined moon shell, Natica lineata. J. fuh . Soc. Taiwan 18, 69-75. HwANo, D . F., CituEit, C. H . and JENG, S . S. (1991b) Tetrodotoxin and derivatives in several spectres of the gastropod Naticidae . Toxicon 29, 1019-1024. HwANo, D . F ., Lu, S. C . and JENo, S . S. (1991 c) Occurrence of tetrodotoxin in the gastropods Rapana rapiformis and R . venosa venosa. Mar. Biol. 111, 65-69 . HwANo, D . F ., LN, L . C . and JENa, S. S. (1992x) Occurrence of tetrodotoxin-related toxins in the gastropod mollusk Niotha clathrata from Taiwan . Nippon Suis. Gakk . 58, 63-67 . HwANo, D. F., LiN, L . C . and JENG, S. S. (19926) Variation and secretion of toxins in gastropod mollusk Niotha clathrata. Toxicon 30, 1189-1194 . HwANa, D. F ., LIN, L. C. and JENo, S. S . (1992c) A new toxin and tetrodotoxin in two species of the gastropod mollusk Nassariidae . Toxicon 30, 41-46. JEoN, J . K., NARITA, H ., NARA, M ., Nooucia, T., MARuyAmA, J. and HAzEmcaTo, K. (1984) Occurrence of tetrodotoxin in a gastropod mollusk, "Araregai" Niotha clathrata. Nippon Suis. Gakk . 50, 2099-2102 .
GTX-3 as Minor Toxin in Niotha clathrata
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NAGAz mrA, Y ., MAauyAmA, J ., Nooucm, T . and HASmmoro, K. (1987) Analysis of paralytic shellfish poison and tetrodotoxin by ion-pairing high performance liquid chromatography. Nippon Suis. Gakk. 30, 85-88 . NAAITA, H ., Nooucm, T., MARuYAuA J., U®A, Y ., HAsmwoTo, K ., WATANABE, Y . and HIDA, K . (1981) Occurrence of tetrodotoxin in a trumpet shell, Charonia sauliae. Nippon Suis. Gakk. 47, 935-941 . NARITA, H ., Noaucm, T ., MAxuyAmA, J., NARA, M . and HAsmmaro, K . (1984) Occurrence of tetrodotoxin-associated substance in a gastropod, "hanamushirogai" Zeuxis siquijorensis . Nippon Suis. Gakk . 50, 8548 . NARrrA, H ., MAsuhom, H ., Mizum, S., Noaucm, T., SAiTo, T., $ir1DA, Y . and HASmsoTo, K . (1989) Anatomical distribution oftetrodotoxin in a trumpet shell, Charonia saullae, along with a comparison of toxin compositions in different tissues. J. Fd Hyg. Soc. Jpn 30, 522-525. Nooucin, T ., MAituyAmA J ., UEDA, Y ., HAsmroro, K. and HARAim, T . (1981) Occurrence of tetrodotoxin in the Japanese ivory shell Babylonia japonica. Nippon Suis. Gakk. 47, 909-913 . Nooucm, T., MAAUYAmA, J ., NAAITA, H . and HAsmioTo, K . (1984) Occurrence of tetrodotoxin in the gastropod mollusk Tutufa lWostoma (frog shell) . Toxicon 22, 219-'226 . OGATA, T ., lsinmARu, T. and KoDAMA, M . (1987) Effect of water temperature and light intensity on growth rate and toxicity change in Protogonyaulaz tamarensis. Mar. Biol. 95, 217-220. OsmiA, Y ., HAsiaAwA, M ., YAsuetoro, T., HALLEoRAw, G . and BLAcaeuRN, S . (1987) Dinofiagellate Gymnodiniam catenatwn as the source of paralytic shellfish toxins in Tasmanian shellfish. Toxicon 23, 1105-1111 . UF.DAY., Nooucm, T., ONouF, Y., KoyAmA, K ., KoNo, M. and HAsmioro, K . (1982) Occurrence of PSP-infested scallops in Ofunato Bay during 1976-1979 and investigation of responsible plankton. Nippon Suis . Gakk . 48, 455-458. YAsusoro, T., Osmuk, Y., Ho&AxA, M. and MryAxom, S . (1981) Occurrence of tetrodotoxin in the ivory shell Babylonie japonica from Wakasa Bay . Nippon Suis. Gakk . 47, 929-934.