- Email: [email protected]
Tetrodotoxin and its analogues in extracts from the toad Atelopus oxyrhynchus (family: Bufonidae)
rox1rar. Vol . 30. No. 11 . pp. 1499-1492, 1992. Printed in Great Britain .
0041-0101/92 $5.00 + .00 1..) 1992 Perpmon Plea Ltd
TETRODOTOXIN AND ITS ANALOGUES IN EXTRACTS FROM THE TOAD ATELOPUS OXYRHYNCHUS (FAMILY: BUFONIDAE) MARI YOTSU-YAMASHITA,' DIETRICH MEHS2*
and TAKESHI
YASUMOTOI
'Faculty of Agriculture, Tohoku University, Tsutsumidori-Amamiya, Aoba-ku, Sendai 981, Japan ; and 2Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt-70, F.R .G. (Received
18
March
1992 ;
accepted 18 June
1992)
D. MESS and T. YAsumoTo . Tetrodotoxin and its analogues in extracts from the toad Atelopus oxyrhynchus (family: Bufonidae) . Toxicon 30, 1489-1492, 1992.-Tetrodotoxin and its analogues, 4epitetrodotoxin and 4,9-anhydrotetrodotoxin, were detected in the toad Atelopus oxyrhynchus by HPLC analysis. The toxin and its analogues were still present in a specimen which lived 3.5 years in captivity. M . YOTSU-YAMASHITA,
toads of the genus Atelopus have been found to contain tetrodotoxin (TTX) and its unique analogues (FUHRMAN, 1986), chiriquitoxin in A. chiriquiensis (KIM et al., 1975 ; YoTsu et al., 1990) and zetekitoxin in A . various zeteki (BROWN et al., 1977). Previously, MESS and SCHMIDT (1989) confirmed that the alcoholic extracts of the Venezuelan toad Atelopus oxyrhynchus contained TTX as proved by derivatization of TTX to the C.9 base . Further analysis of the toxin and its analogues was performed using a fluorometric HPLC analyzer with high sensitivity (YoTsu and YAsumoTo, 1989). Atelopus oxyrhynchus specimen (2) which died 2 months after collection in a mountain forest near La Azulita, Merida Prov ., Venezuela, in August 1987 (sample 1) and a specimen (1) which died after 3 .5 years in captivity (sample 2) were extracted (whole body) with 80% ethanol acidified to pH 2.0 with HC1 . The extracts were concentrated in vacuo and evaporated to dryness under reduced pressure. Toxicity was determined using the mouse-bioassay (male mice, ddY strain, 15-20 g, i.p. injection) standardized for TTX (YASUMOTO, 1991) and expressed in mouse units (MU). One unit is equivalent to 0.22 hg TROPICAL
TTX.
HPLC-analysis was performed on a Develosil ODS-5 column, using post-column reaction with sodium hydroxide and fluorescence detection as described previously (YoTsu and YASUMOTO, 1989). The detection limit was 6.6 ng TTX. To clean up the extracts the following procedures were carried out using activated charcoal and a cation exchange gel (Hitachi 3011 c) packed in glass pipettes: 8 MU of each sample was dissolved in distilled water (100,u1) and applied to 150 yl charcoal or 50,ul gel, respectively ; each column was washed with 200,ul water and eluted with 500,u1 1 % acetic acid in 20% Author to whom correspondence should be addressed . 1489
1490
Short Communications
2 3
2
E
D 4
2
I5
IV 0
9
VN 16
A'A-M 0
6
16 min
FIG. l. HPLC of ALCOHOLIC ExTItAcis FROM: Arelopus OXYMynchus ON A DEVELOSIL ODS-5 COLUMN
(0.46 x 25 cm). The mobile phase consisted of 0.06 M ammonium trifluoroacetate, 0.05 M NH4 OAc and 3% McCN (pH 5.0). Flow rate, 0.5 ml/min . Post-column reaction was performed with 2.8 N NaOH in 0.5 mm x 10 m stainless coil at 105°C; fluorescence was measured at 510 nm (excitation 365 nm). A, Standard mixture: (1) tetrodonic acid, (2) tetrodotoxin (TTX), (3) 6-ep=, (4) 4-ep=, (5) 4,9-anhydroTTX ; B, Sample 1 (from animals 2 months after collection) after clean-up with activated charcoal; and C, with Hitachi gel 3011 c; D, Sample 2 (animal after 3.5 years in captivity) after clean-up with activated charcoal, and E, with 3011c gel .
ethanol from charcoal or with 100 pl 0.5 M acetic acid from 3011c gel, respectively . Chromatography on the cation exchange gel was performed by centrifugation of the pipette (15,000 rpm, 20 sec) to allow the solvent to flow through the gel. Eluates were
Short Communications
1491
TABLE 1 . REsuLTs oP INousE-BRiAmy AND HPLC-ANALYSES oP TTX IN ExrRAcrs oP Atelopus oxyrhynchus ExPREssim IN MousE uNrrs (MU)
Sample I Toxicity (total) Clean-up with charcoal Amount applied Amount eluted Recovery (%) Clean-up with 3011c gel Amount applied Amount eluted Recovery (%) Sample 2 Toxicity (total Clean-up with charcoal Amount applied Amount eluted Recovery (%) Clean-up with 3011c gel Amount applied Amount eluted Recovery (%)
Mouse-bioassay (MU)
HPLC (MU)
50/g
nd'
8 .0 3 .7 47 .0
3 .4 42.0
8 .0 3 .8 47 .0
3 .8 47.0
161 .2/g
144/g
8 .0 4 .0 50 .0
3 .4 45 .0
8 .0 2 .6 32 .0
2.7 34.0
'nd: not determined .
evaporated (charcoal) or used without further treatment (3011c gel) for mouse-bioassay and HPLC . Chromatograms of both sample 1 (from two animals, 2 months after collection) and sample 2 (from one animal, 3.5 years in captivity) revealed peaks corresponding to TTX and TTX-analogues, 4-epi= and 4,9-anhydroTTX, after clean-up procedure using charcoal as well as 301 lc gel (Fig. 1). This indicates that TTX and its analogues were kept after 3.5 years' captivity. The absence of 6-eptTTX, a TTX-analogue found in newts (YASUMoTO et al., 1988) and puffer-fish (ENDO et al., 1988) is suggested in A. oxyrhynchus . The peaks appearing between those corresponding to TTX and 4ep:TTX in the chromatograms of the eluates from charcoal of both samples were suspected to correspond to 6-ept1TX. However, the retention time of those peaks were slightly different from that of 6-epiTI'X. This unknown peak was effectively removed by clean-up with 3011 c gel, while other TTX analogues were recovered in the eluates. The good correlation between the toxicities determined by bioassay and those by HPLC ruled out the presence of chiriquitoxin. Although this toxin is low sensitive in the HPLC system, it should be detected by a discrepancy between the two assay methods. Toxicity values of both samples, 50 and 161 .2 MU/g, respectively, are lower than those estimated for Atelopus varius (100-1200 MU/g) and A. chiriquiensis (350 MU/g; KIM et al., 1975). But it is interesting to note that Atelopus oxyrhynchus is able to keep a high level of toxicity living more than 3 years in captivity and in an artificial environment. It is well established that TTX in marine animals is of bacterial origin (YAsumoTO et al., 1986; Smminu et al., 1987). In terrestrial animals the origin and biosynthetic pathway of TTX is still not known, since in feeding experiments on newts incorporation of several TTX-precursors did not occur (SHImzu and KOBAYASHI, 1983).
1492
Short Communications REFERENCES
BRowN, G . B., Kim, Y. H ., KuNTzEL, H . and Mostwt, H. S. (1977) Chemistry and pharmacology of skin toxins from the frog Atelopus zeteki (atelopidtoxin : zetekitoxin) . Toxicon 15, 115-128 . ENDG, A., KHoRA, S . S ., NAGKj, H . and YAsuetoro, T. (1988) Isolation of 11-nortetrodotoxin-6 (R)-ol and other tetrodotoxin derivatives from the puffer Fugu niphobles . Tetrahedron Lett. 33, 4127-4128 . FuHxmAN, F. A. (1986) Tetrodotoxin, tarichatoxin, and chiriquitoxin : historical perspectives . Ann. N. Y . Acad. Sci. 479, 1-14 . Kim, Y. H ., BROWN, G . B., MosHER, H . S . and FutflutAN, F. A . (1975) Tetrodotoxin : occurrence in atelopid frogs of Costa Rica. Science 189, 151-152. M®s, D . uND SctnDT, K . (1989) Occurrence of tetrodotoxin in the frog Atelopus oxyrhynchus . Toxicon 27, 819-822. SHihow, U ., NoGucm, T., HWANG, D . F., SHIDA, Y . and HAstnmoro, K. (1987) Marine bacteria which produce tetrodotoxin. Appl. Envir. Microblol. 53, 1714-1715 . SHnazu, Y . and KoRAYAsm, M . (1983) Apparent lack of tetrodotoxin biosynthesis in captured Taricha torosa and Taricha gramdosa . Chem . Pharm . Bull. 31, 3625-3631 . YASUmoro, T . (1991) Puffer toxin . In : The Manualfor the Methods of Food Sanitation Tests, Chemistry, pp. 296300 . Tokyo : Japan Food Hygienic Association . YAstmoTo, T., YAsumuRA, D., YoTsu, M ., MtctnsHrTA, T., ENDo, A. and KoTAKj, Y . (1986) Bacterial production of tetrodotoxin and anhydrotetrodotoxin . Agric. Biol. Chem . 50, 793-795 . YAsumoTo, T ., YOTSU, M., MURATA, M . and NAom, H. (1988) New tetrodotoxin analogues from the newt Cynops ensicauda. !. Am. chem. Soc. 110, 2344-2345. YoTsu, M . and YASUSfoTo, T . (1989) An improved tetrodotoxin analyser. Agric. Mol. Chem . 53, 893-895. YoTsu, M ., YAsumoTo, T., Kim, Y . H ., NAoKi, H . and KAo, C . Y. (1990) The structure of chiriquitoxin from the Costa Rican frog Atelopus chiriquiensis . Tetrahedron Lett. 31, 3187-3190.
0041-0101/92 $5.00 + .00 1..) 1992 Perpmon Plea Ltd
TETRODOTOXIN AND ITS ANALOGUES IN EXTRACTS FROM THE TOAD ATELOPUS OXYRHYNCHUS (FAMILY: BUFONIDAE) MARI YOTSU-YAMASHITA,' DIETRICH MEHS2*
and TAKESHI
YASUMOTOI
'Faculty of Agriculture, Tohoku University, Tsutsumidori-Amamiya, Aoba-ku, Sendai 981, Japan ; and 2Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt-70, F.R .G. (Received
18
March
1992 ;
accepted 18 June
1992)
D. MESS and T. YAsumoTo . Tetrodotoxin and its analogues in extracts from the toad Atelopus oxyrhynchus (family: Bufonidae) . Toxicon 30, 1489-1492, 1992.-Tetrodotoxin and its analogues, 4epitetrodotoxin and 4,9-anhydrotetrodotoxin, were detected in the toad Atelopus oxyrhynchus by HPLC analysis. The toxin and its analogues were still present in a specimen which lived 3.5 years in captivity. M . YOTSU-YAMASHITA,
toads of the genus Atelopus have been found to contain tetrodotoxin (TTX) and its unique analogues (FUHRMAN, 1986), chiriquitoxin in A. chiriquiensis (KIM et al., 1975 ; YoTsu et al., 1990) and zetekitoxin in A . various zeteki (BROWN et al., 1977). Previously, MESS and SCHMIDT (1989) confirmed that the alcoholic extracts of the Venezuelan toad Atelopus oxyrhynchus contained TTX as proved by derivatization of TTX to the C.9 base . Further analysis of the toxin and its analogues was performed using a fluorometric HPLC analyzer with high sensitivity (YoTsu and YAsumoTo, 1989). Atelopus oxyrhynchus specimen (2) which died 2 months after collection in a mountain forest near La Azulita, Merida Prov ., Venezuela, in August 1987 (sample 1) and a specimen (1) which died after 3 .5 years in captivity (sample 2) were extracted (whole body) with 80% ethanol acidified to pH 2.0 with HC1 . The extracts were concentrated in vacuo and evaporated to dryness under reduced pressure. Toxicity was determined using the mouse-bioassay (male mice, ddY strain, 15-20 g, i.p. injection) standardized for TTX (YASUMOTO, 1991) and expressed in mouse units (MU). One unit is equivalent to 0.22 hg TROPICAL
TTX.
HPLC-analysis was performed on a Develosil ODS-5 column, using post-column reaction with sodium hydroxide and fluorescence detection as described previously (YoTsu and YASUMOTO, 1989). The detection limit was 6.6 ng TTX. To clean up the extracts the following procedures were carried out using activated charcoal and a cation exchange gel (Hitachi 3011 c) packed in glass pipettes: 8 MU of each sample was dissolved in distilled water (100,u1) and applied to 150 yl charcoal or 50,ul gel, respectively ; each column was washed with 200,ul water and eluted with 500,u1 1 % acetic acid in 20% Author to whom correspondence should be addressed . 1489
1490
Short Communications
2 3
2
E
D 4
2
I5
IV 0
9
VN 16
A'A-M 0
6
16 min
FIG. l. HPLC of ALCOHOLIC ExTItAcis FROM: Arelopus OXYMynchus ON A DEVELOSIL ODS-5 COLUMN
(0.46 x 25 cm). The mobile phase consisted of 0.06 M ammonium trifluoroacetate, 0.05 M NH4 OAc and 3% McCN (pH 5.0). Flow rate, 0.5 ml/min . Post-column reaction was performed with 2.8 N NaOH in 0.5 mm x 10 m stainless coil at 105°C; fluorescence was measured at 510 nm (excitation 365 nm). A, Standard mixture: (1) tetrodonic acid, (2) tetrodotoxin (TTX), (3) 6-ep=, (4) 4-ep=, (5) 4,9-anhydroTTX ; B, Sample 1 (from animals 2 months after collection) after clean-up with activated charcoal; and C, with Hitachi gel 3011 c; D, Sample 2 (animal after 3.5 years in captivity) after clean-up with activated charcoal, and E, with 3011c gel .
ethanol from charcoal or with 100 pl 0.5 M acetic acid from 3011c gel, respectively . Chromatography on the cation exchange gel was performed by centrifugation of the pipette (15,000 rpm, 20 sec) to allow the solvent to flow through the gel. Eluates were
Short Communications
1491
TABLE 1 . REsuLTs oP INousE-BRiAmy AND HPLC-ANALYSES oP TTX IN ExrRAcrs oP Atelopus oxyrhynchus ExPREssim IN MousE uNrrs (MU)
Sample I Toxicity (total) Clean-up with charcoal Amount applied Amount eluted Recovery (%) Clean-up with 3011c gel Amount applied Amount eluted Recovery (%) Sample 2 Toxicity (total Clean-up with charcoal Amount applied Amount eluted Recovery (%) Clean-up with 3011c gel Amount applied Amount eluted Recovery (%)
Mouse-bioassay (MU)
HPLC (MU)
50/g
nd'
8 .0 3 .7 47 .0
3 .4 42.0
8 .0 3 .8 47 .0
3 .8 47.0
161 .2/g
144/g
8 .0 4 .0 50 .0
3 .4 45 .0
8 .0 2 .6 32 .0
2.7 34.0
'nd: not determined .
evaporated (charcoal) or used without further treatment (3011c gel) for mouse-bioassay and HPLC . Chromatograms of both sample 1 (from two animals, 2 months after collection) and sample 2 (from one animal, 3.5 years in captivity) revealed peaks corresponding to TTX and TTX-analogues, 4-epi= and 4,9-anhydroTTX, after clean-up procedure using charcoal as well as 301 lc gel (Fig. 1). This indicates that TTX and its analogues were kept after 3.5 years' captivity. The absence of 6-eptTTX, a TTX-analogue found in newts (YASUMoTO et al., 1988) and puffer-fish (ENDO et al., 1988) is suggested in A. oxyrhynchus . The peaks appearing between those corresponding to TTX and 4ep:TTX in the chromatograms of the eluates from charcoal of both samples were suspected to correspond to 6-ept1TX. However, the retention time of those peaks were slightly different from that of 6-epiTI'X. This unknown peak was effectively removed by clean-up with 3011 c gel, while other TTX analogues were recovered in the eluates. The good correlation between the toxicities determined by bioassay and those by HPLC ruled out the presence of chiriquitoxin. Although this toxin is low sensitive in the HPLC system, it should be detected by a discrepancy between the two assay methods. Toxicity values of both samples, 50 and 161 .2 MU/g, respectively, are lower than those estimated for Atelopus varius (100-1200 MU/g) and A. chiriquiensis (350 MU/g; KIM et al., 1975). But it is interesting to note that Atelopus oxyrhynchus is able to keep a high level of toxicity living more than 3 years in captivity and in an artificial environment. It is well established that TTX in marine animals is of bacterial origin (YAsumoTO et al., 1986; Smminu et al., 1987). In terrestrial animals the origin and biosynthetic pathway of TTX is still not known, since in feeding experiments on newts incorporation of several TTX-precursors did not occur (SHImzu and KOBAYASHI, 1983).
1492
Short Communications REFERENCES
BRowN, G . B., Kim, Y. H ., KuNTzEL, H . and Mostwt, H. S. (1977) Chemistry and pharmacology of skin toxins from the frog Atelopus zeteki (atelopidtoxin : zetekitoxin) . Toxicon 15, 115-128 . ENDG, A., KHoRA, S . S ., NAGKj, H . and YAsuetoro, T. (1988) Isolation of 11-nortetrodotoxin-6 (R)-ol and other tetrodotoxin derivatives from the puffer Fugu niphobles . Tetrahedron Lett. 33, 4127-4128 . FuHxmAN, F. A. (1986) Tetrodotoxin, tarichatoxin, and chiriquitoxin : historical perspectives . Ann. N. Y . Acad. Sci. 479, 1-14 . Kim, Y. H ., BROWN, G . B., MosHER, H . S . and FutflutAN, F. A . (1975) Tetrodotoxin : occurrence in atelopid frogs of Costa Rica. Science 189, 151-152. M®s, D . uND SctnDT, K . (1989) Occurrence of tetrodotoxin in the frog Atelopus oxyrhynchus . Toxicon 27, 819-822. SHihow, U ., NoGucm, T., HWANG, D . F., SHIDA, Y . and HAstnmoro, K. (1987) Marine bacteria which produce tetrodotoxin. Appl. Envir. Microblol. 53, 1714-1715 . SHnazu, Y . and KoRAYAsm, M . (1983) Apparent lack of tetrodotoxin biosynthesis in captured Taricha torosa and Taricha gramdosa . Chem . Pharm . Bull. 31, 3625-3631 . YASUmoro, T . (1991) Puffer toxin . In : The Manualfor the Methods of Food Sanitation Tests, Chemistry, pp. 296300 . Tokyo : Japan Food Hygienic Association . YAstmoTo, T., YAsumuRA, D., YoTsu, M ., MtctnsHrTA, T., ENDo, A. and KoTAKj, Y . (1986) Bacterial production of tetrodotoxin and anhydrotetrodotoxin . Agric. Biol. Chem . 50, 793-795 . YAsumoTo, T ., YOTSU, M., MURATA, M . and NAom, H. (1988) New tetrodotoxin analogues from the newt Cynops ensicauda. !. Am. chem. Soc. 110, 2344-2345. YoTsu, M . and YASUSfoTo, T . (1989) An improved tetrodotoxin analyser. Agric. Mol. Chem . 53, 893-895. YoTsu, M ., YAsumoTo, T., Kim, Y . H ., NAoKi, H . and KAo, C . Y. (1990) The structure of chiriquitoxin from the Costa Rican frog Atelopus chiriquiensis . Tetrahedron Lett. 31, 3187-3190.