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The levels of tetrodotoxin and its analogue 6-epitetrodotoxin in the red-spotted newt, Notophthalmus viridescens
Toxicon 39 (2001) 1261±1263
Short communication
www.elsevier.com/locate/toxicon
The levels of tetrodotoxin and its analogue 6-epitetrodotoxin in the red-spotted newt, Notophthalmus viridescens Mari Yotsu-Yamashita a, Dietrich Mebs b,* a
Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555 Japan b Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt, Germany Received 20 June 2000; accepted 8 December 2000
Abstract Tetrodotoxin (TTX) and its analogue 6-epiTTX were detected in 11±12 specimens of the red-spotted newt, Notophthalmus viridescens, by a post-column ¯uorescent-HPLC system and by LC/MS in selected ion monitoring mode. TTX levels varied considerably among individuals from low (less than 0.15 mg TTX/g newt) to high concentrations (23.5 mg TTX/g newt), while 6-epiTTX was found to be a minor constituent in all specimens. q 2001 Elsevier Science Ltd. All rights reserved.
Tetrodotoxin (TTX) occurs in a wide range of marine and terrestrial animals (Yasumoto and Yotsu-Yamashita, 1996) including newts and salamanders, where the toxin and its analogues 6-epi TTX and/or 11-deoxyTTX (Yasumoto et al., 1988; Mebs et al., 1995) have been detected in species of Ambystoma, Cynops, Notophthalmus, Taricha (Hani®n et al., 1999) and Triturus, and only TTX has been detected in Paramesotriton (Yotsu et al., 1990). Since Brodie et al. (1974); Levenson and Woodhull (1979) had already described the occurrence of a tetrodotoxin-like substance in the red-spotted newt, Notophthalmus viridescens from the eastern part of North-America, and Yotsu et al. (1990) identi®ed TTX and 6-epiTTX in the pooled sample by 1HNMR spectroscopy and thin layer chromatography, we present further analytical data on 12 specimens of this newt. Twelve adult specimens (aquatic form) of N. viridescens were collected in October 1999 in the main pond of the Mountain Lake Biological Station of the University of Virginia (Pembroke, VA 24136, USA) at an altitude of 1160 m. The newts were killed by freezing and placed in 70 vol.% methanol containing 0.1 vol.% acetic acid. The alcoholic extracts were evaporated to dryness at 458C in a stream of nitrogen. Each sample was dissolved in 0.05 M acetic acid (0.5 ml/g newt), centrifuged and the supernatant was puri®ed by ultra®ltration (Millipore, * Corresponding author. Tel.: 149-69-6301-7418; fax: 149-696301-5882. E-mail address: [email protected] (D. Mebs).
Ultrafree-MC UFC3LTK00). The clear ®ltrate was 50 times diluted with 0.05 M acetic acid and 10 ml was applied to a post column HPLC-¯uorescent detection (HPLC-FLD) (Yasumoto and Michishita, 1985; Shoji et al., in press) using a Develosil column (C30-UG-5, 0.46 i.d. £ 25 cm, Nomura Chemical, Seto, Japan), an aqueous solution containing 1 vol.% acetonitrile, 30 mM ammonium hepta¯uorobutyrate and 10 mM ammonium formate buffer (pH 5.0) for the mobile phase (48C) at a ¯ow rate of 0.4 ml/min., 4 N NaOH for post-column reaction, and a JASCO FP-920 ¯uorescent monitor for detection (Ex. 365, Em 510 nm). LC/MS in selective ion monitoring mode (SIM) was also used for the search of other analogues. For LC/MS, the same column and an aqueous solution containing 1 vol.% acetonitrile, 20 mM ammonium hepta¯uorobutyrate and 10 mM ammonium formate buffer (pH 4.0) were used at 158C at a ¯ow rate of 0.4 ml/min, and 2.5 ml of sample solution was applied (Shoji et al., in press). Toxicity of the extracts was determined using the mouse bioassay (male mice, ddY strain, 15±20 g, i.p. injection) standardized for TTX (Yasumoto, 1991). The HPLC-FLD chromatogram and the SIM detected mass chromatogram on LC/MS for the specimen 4 are shown in Fig. 1 for representative. The levels of TTX and of its analogue 6-epiTTX in the methanolic extracts of 12 newts and the toxicities are summarised in Table 1. Although the TTX-content was highly variable, in one specimen (7) the toxin was not even detectable (detection
0041-0101/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0041-010 1(00)00263-4
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M. Yotsu-Yamashita, D. Mebs / Toxicon 39 (2001) 1261±1263
Fig. 1. HPLC-FLD chromatograms (a, b) and LC-MS mass chromatograms in SIM mode at m/z 290, 302, 304, 320 and a reconstructed ion current (RIC) (c,d) for standard TTX mixture (a,c) and specimen 4 of N. viridescens (b,d).See text for chromatographic conditions. (a) A mixture of TTX (5.5 ng), 6-epiTTX (0.075 ng), 4-epiTTX (1.2 ng), and 4,9-anhydroTTX (1.3 ng). (b) 10 ml of 50-fold diluted sample solution (see text for preparation).(c) Mixture of TTX (64 ng), 6-epiTTX (16 ng), 11-norTTX-6(S)-ol (20 ng), 4-epiTTX (9.6 ng), 11-deoxyTTX (27 ng), 11-norTTX-6(R)-ol (5.8 ng), 4,9-anhydroTTX (24 ng) and 5-deoxyTTX (24 ng). (d) 2.5 ml of sample solution.
limit of TTX on HPLC-FLD was 0.15 ng), 6-epiTTX was present in all newt extracts as a minor constituent only. The toxicities estimated by HPLC-FLD were approximately 40± 90% of those determined by bioassay, suggesting the presence of undetectable toxins in the extracts. TTX, 6epiTTX, 4-epiTTX (m/z 320), and 4,9-anhydroTTX (m/z 302) were also detected by LC/MS in SIM mode, while 5deoxyTTX (Yotsu-Yamashita et al., 1999), 11-deoxyTTX (m/z 304), 11-norTTX-6(S)-ol (m/z 290) (Yotsu et al., 1992), and 11-norTTX-6(R)-ol (m/z 290) (Endo et al., 1988), which were found in puffer ®sh, were less than their detection limits (0.2 ng) in all specimens.
These data further con®rm the presence of TTX and of its analogue 6-epiTTX in the newt Notophthalmus viridescens and demonstrate a considerable individual variability in toxin concentrations, ranging from less than 0.15±23.5 mg TTX/g. Levenson and Woodhull (1979) had estimated a toxin concentration of 70 MU/g of newt, which is in the same order of magnitude as assayed in the present study: 51±139 MU/g (except two values ,50 MU/g). Like in other amphibian species including frogs, toads, newts and salamanders, TTX may provide protection from predators, acting as a warning signal and as deterrent.
M. Yotsu-Yamashita, D. Mebs / Toxicon 39 (2001) 1261±1263
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Table 1 Results of HPLC analysis and mouse bioassay of extracts from red-spotted newts, Notophthalmus viridescens Specimen no./sex (m/f) a
1/m 2/m 3/f 4/f 5/m 6/f 7/m 8/f 9/m 10/m 11/f 12/m a
Body weight (g)
4.0 2.0 3.2 2.6 3.6 3.3 3.5 3.3 4.0 4.0 3.0 4.7
TTX (mg/g)
20.5 5.3 11.5 23.5 18.0 20.8 ,0.15 8.5 15.3 4.7 21.8 16.3
6-epiTTX (mg/g)
1.08 0.50 0.33 0.80 0.88 0.68 0.05 0.48 0.68 0.20 0.45 0.55
Toxicity (MU/g) Bioassay
(HPLC equiv.)
126 ,50 89 139 134 123 ,50 75 89 51 111 115
(92.9) (24.1) (52.5) (106.6) (81.7) (93.8) (,0.5) (38.8) (69.1) (21.4) (98.4) (73.41)
Male/female.
Acknowledgements We would like to thank Dr H.M. Wilbur, Mountain Lake Biological Station, University of Virginia, for providing the opportunity to collect the newts. This work was supported in part by a grant of Deutsche Forschungsgemeinschaft to D.M., by Grants-in Aid from the Ministry of Education, Science, Sports and Culture of Japan (12045210), from the Naito Foundation and the Fujisawa Foundation to M.Y.Y.
References Brodie, E.D., Hensel, J.L., Johnson, J.A., 1974. Toxicity of the urodele amphibians Taricha, Notophthalmus, Cynops and Paramesotriton (Salamandridae). Copeia, 506±511. Endo, A., Khora, S.S., Murata, M., Yasumoto, T., 1988. Isolation of 11-nortetrodotoxin-6(R)-ol and other tetrodotoxin derivatives from the puffer Fugu niphobles. Tetrahedron Lett. 29, 4127± 4128. Hani®n, C.T., Yotsu-Yamashita, M., Yasumoto, T., Broodie III, E.D., Broodie Jr, E.D., 1999. Toxicity of dangerous prey: variation of tetrodotoxin levels within and among populations of the newt Taricha granulosa. J. Chem. Ecology 25, 2161±2175. Levenson, C.H., Woodhull, A.M., 1979. The occurrence of a tetrodotoxin-like substance in the red-spotted newt, Notophthalmus viridescens. Toxicon 17, 184±187. Mebs, D., Yotsu-Yamashita, M., Yasumoto, T., LoÈtters, S., SchluÈ-
ter, A., 1995. Further report of the occurrence of tetrodotoxin in Atelopus species (family: Bufonidae). Toxicon 33, 246±249. Shoji, Y., Yotsu-Yamashita, M., Miyazawa, T., Yasumoto, T., 2001. Electrospray ionization mass spectrometry of tetrodotoxin and its analogs: liquid chromatography/mass spectrometry, tandem mass spectrometry, and liquid chromatography/tandem mass spectrometry. Anal. Biochem. in press. Yasumoto, T., Michishita, T., 1985. Fluorometric determination of tetrodotoxin by high performance liquid chromatography. Agric. Biol. Chem. 49, 3077±3080. Yasumoto, T., Yotsu, M., Murata, M., Naoki, H., 1988. New tetrodotoxin analogues from the newt Cynops ensicauda. J. Am. Chem. Soc. 110, 2344±2345. Yasumoto, T., 1991. Puffer®sh toxin. In: Bureau of Environmental Health. Ministry of Health and Welfare, Japan (Ed.). The Manual for the Methods of Food Sanitation Tests. Food Hygenic Association, Tokyo, pp. 296±300. Yasumoto, T., Yotsu-Yamashita, M., 1996. Chemical and etiological studies on tetrodotoxin and its analogs. J. Toxicol. Toxin Reviews 15, 81±90. Yotsu, M., Iorizzi, M., Yasumoto, T., 1990. Distribution of tetrodotoxin, 6-epitetrodotoxin and 11-deoxytetrodotoxin in newts. Toxicon 28, 238±241. Yotsu, M., Hayashi, Y., Khora, S.S., Sato, S., Yasumoto, T., 1992. Isolation and structural assignment of 11-nortetrodotoxin-6(S)ol from the puffer Arothron nigropunctatus. Biosci. Biotech. Biochem. 56, 370±371. Yotsu-Yamashita, M., Schimmele, B., Yasumoto, T., 1999. Isolation and structural assignment of 5-deoxytetrodotoxin from the puffer ®sh Fugu poecilonotus. Biosci. Biotechnol. Biochem. 63, 961±963.
Short communication
www.elsevier.com/locate/toxicon
The levels of tetrodotoxin and its analogue 6-epitetrodotoxin in the red-spotted newt, Notophthalmus viridescens Mari Yotsu-Yamashita a, Dietrich Mebs b,* a
Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555 Japan b Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt, Germany Received 20 June 2000; accepted 8 December 2000
Abstract Tetrodotoxin (TTX) and its analogue 6-epiTTX were detected in 11±12 specimens of the red-spotted newt, Notophthalmus viridescens, by a post-column ¯uorescent-HPLC system and by LC/MS in selected ion monitoring mode. TTX levels varied considerably among individuals from low (less than 0.15 mg TTX/g newt) to high concentrations (23.5 mg TTX/g newt), while 6-epiTTX was found to be a minor constituent in all specimens. q 2001 Elsevier Science Ltd. All rights reserved.
Tetrodotoxin (TTX) occurs in a wide range of marine and terrestrial animals (Yasumoto and Yotsu-Yamashita, 1996) including newts and salamanders, where the toxin and its analogues 6-epi TTX and/or 11-deoxyTTX (Yasumoto et al., 1988; Mebs et al., 1995) have been detected in species of Ambystoma, Cynops, Notophthalmus, Taricha (Hani®n et al., 1999) and Triturus, and only TTX has been detected in Paramesotriton (Yotsu et al., 1990). Since Brodie et al. (1974); Levenson and Woodhull (1979) had already described the occurrence of a tetrodotoxin-like substance in the red-spotted newt, Notophthalmus viridescens from the eastern part of North-America, and Yotsu et al. (1990) identi®ed TTX and 6-epiTTX in the pooled sample by 1HNMR spectroscopy and thin layer chromatography, we present further analytical data on 12 specimens of this newt. Twelve adult specimens (aquatic form) of N. viridescens were collected in October 1999 in the main pond of the Mountain Lake Biological Station of the University of Virginia (Pembroke, VA 24136, USA) at an altitude of 1160 m. The newts were killed by freezing and placed in 70 vol.% methanol containing 0.1 vol.% acetic acid. The alcoholic extracts were evaporated to dryness at 458C in a stream of nitrogen. Each sample was dissolved in 0.05 M acetic acid (0.5 ml/g newt), centrifuged and the supernatant was puri®ed by ultra®ltration (Millipore, * Corresponding author. Tel.: 149-69-6301-7418; fax: 149-696301-5882. E-mail address: [email protected] (D. Mebs).
Ultrafree-MC UFC3LTK00). The clear ®ltrate was 50 times diluted with 0.05 M acetic acid and 10 ml was applied to a post column HPLC-¯uorescent detection (HPLC-FLD) (Yasumoto and Michishita, 1985; Shoji et al., in press) using a Develosil column (C30-UG-5, 0.46 i.d. £ 25 cm, Nomura Chemical, Seto, Japan), an aqueous solution containing 1 vol.% acetonitrile, 30 mM ammonium hepta¯uorobutyrate and 10 mM ammonium formate buffer (pH 5.0) for the mobile phase (48C) at a ¯ow rate of 0.4 ml/min., 4 N NaOH for post-column reaction, and a JASCO FP-920 ¯uorescent monitor for detection (Ex. 365, Em 510 nm). LC/MS in selective ion monitoring mode (SIM) was also used for the search of other analogues. For LC/MS, the same column and an aqueous solution containing 1 vol.% acetonitrile, 20 mM ammonium hepta¯uorobutyrate and 10 mM ammonium formate buffer (pH 4.0) were used at 158C at a ¯ow rate of 0.4 ml/min, and 2.5 ml of sample solution was applied (Shoji et al., in press). Toxicity of the extracts was determined using the mouse bioassay (male mice, ddY strain, 15±20 g, i.p. injection) standardized for TTX (Yasumoto, 1991). The HPLC-FLD chromatogram and the SIM detected mass chromatogram on LC/MS for the specimen 4 are shown in Fig. 1 for representative. The levels of TTX and of its analogue 6-epiTTX in the methanolic extracts of 12 newts and the toxicities are summarised in Table 1. Although the TTX-content was highly variable, in one specimen (7) the toxin was not even detectable (detection
0041-0101/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0041-010 1(00)00263-4
1262
M. Yotsu-Yamashita, D. Mebs / Toxicon 39 (2001) 1261±1263
Fig. 1. HPLC-FLD chromatograms (a, b) and LC-MS mass chromatograms in SIM mode at m/z 290, 302, 304, 320 and a reconstructed ion current (RIC) (c,d) for standard TTX mixture (a,c) and specimen 4 of N. viridescens (b,d).See text for chromatographic conditions. (a) A mixture of TTX (5.5 ng), 6-epiTTX (0.075 ng), 4-epiTTX (1.2 ng), and 4,9-anhydroTTX (1.3 ng). (b) 10 ml of 50-fold diluted sample solution (see text for preparation).(c) Mixture of TTX (64 ng), 6-epiTTX (16 ng), 11-norTTX-6(S)-ol (20 ng), 4-epiTTX (9.6 ng), 11-deoxyTTX (27 ng), 11-norTTX-6(R)-ol (5.8 ng), 4,9-anhydroTTX (24 ng) and 5-deoxyTTX (24 ng). (d) 2.5 ml of sample solution.
limit of TTX on HPLC-FLD was 0.15 ng), 6-epiTTX was present in all newt extracts as a minor constituent only. The toxicities estimated by HPLC-FLD were approximately 40± 90% of those determined by bioassay, suggesting the presence of undetectable toxins in the extracts. TTX, 6epiTTX, 4-epiTTX (m/z 320), and 4,9-anhydroTTX (m/z 302) were also detected by LC/MS in SIM mode, while 5deoxyTTX (Yotsu-Yamashita et al., 1999), 11-deoxyTTX (m/z 304), 11-norTTX-6(S)-ol (m/z 290) (Yotsu et al., 1992), and 11-norTTX-6(R)-ol (m/z 290) (Endo et al., 1988), which were found in puffer ®sh, were less than their detection limits (0.2 ng) in all specimens.
These data further con®rm the presence of TTX and of its analogue 6-epiTTX in the newt Notophthalmus viridescens and demonstrate a considerable individual variability in toxin concentrations, ranging from less than 0.15±23.5 mg TTX/g. Levenson and Woodhull (1979) had estimated a toxin concentration of 70 MU/g of newt, which is in the same order of magnitude as assayed in the present study: 51±139 MU/g (except two values ,50 MU/g). Like in other amphibian species including frogs, toads, newts and salamanders, TTX may provide protection from predators, acting as a warning signal and as deterrent.
M. Yotsu-Yamashita, D. Mebs / Toxicon 39 (2001) 1261±1263
1263
Table 1 Results of HPLC analysis and mouse bioassay of extracts from red-spotted newts, Notophthalmus viridescens Specimen no./sex (m/f) a
1/m 2/m 3/f 4/f 5/m 6/f 7/m 8/f 9/m 10/m 11/f 12/m a
Body weight (g)
4.0 2.0 3.2 2.6 3.6 3.3 3.5 3.3 4.0 4.0 3.0 4.7
TTX (mg/g)
20.5 5.3 11.5 23.5 18.0 20.8 ,0.15 8.5 15.3 4.7 21.8 16.3
6-epiTTX (mg/g)
1.08 0.50 0.33 0.80 0.88 0.68 0.05 0.48 0.68 0.20 0.45 0.55
Toxicity (MU/g) Bioassay
(HPLC equiv.)
126 ,50 89 139 134 123 ,50 75 89 51 111 115
(92.9) (24.1) (52.5) (106.6) (81.7) (93.8) (,0.5) (38.8) (69.1) (21.4) (98.4) (73.41)
Male/female.
Acknowledgements We would like to thank Dr H.M. Wilbur, Mountain Lake Biological Station, University of Virginia, for providing the opportunity to collect the newts. This work was supported in part by a grant of Deutsche Forschungsgemeinschaft to D.M., by Grants-in Aid from the Ministry of Education, Science, Sports and Culture of Japan (12045210), from the Naito Foundation and the Fujisawa Foundation to M.Y.Y.
References Brodie, E.D., Hensel, J.L., Johnson, J.A., 1974. Toxicity of the urodele amphibians Taricha, Notophthalmus, Cynops and Paramesotriton (Salamandridae). Copeia, 506±511. Endo, A., Khora, S.S., Murata, M., Yasumoto, T., 1988. Isolation of 11-nortetrodotoxin-6(R)-ol and other tetrodotoxin derivatives from the puffer Fugu niphobles. Tetrahedron Lett. 29, 4127± 4128. Hani®n, C.T., Yotsu-Yamashita, M., Yasumoto, T., Broodie III, E.D., Broodie Jr, E.D., 1999. Toxicity of dangerous prey: variation of tetrodotoxin levels within and among populations of the newt Taricha granulosa. J. Chem. Ecology 25, 2161±2175. Levenson, C.H., Woodhull, A.M., 1979. The occurrence of a tetrodotoxin-like substance in the red-spotted newt, Notophthalmus viridescens. Toxicon 17, 184±187. Mebs, D., Yotsu-Yamashita, M., Yasumoto, T., LoÈtters, S., SchluÈ-
ter, A., 1995. Further report of the occurrence of tetrodotoxin in Atelopus species (family: Bufonidae). Toxicon 33, 246±249. Shoji, Y., Yotsu-Yamashita, M., Miyazawa, T., Yasumoto, T., 2001. Electrospray ionization mass spectrometry of tetrodotoxin and its analogs: liquid chromatography/mass spectrometry, tandem mass spectrometry, and liquid chromatography/tandem mass spectrometry. Anal. Biochem. in press. Yasumoto, T., Michishita, T., 1985. Fluorometric determination of tetrodotoxin by high performance liquid chromatography. Agric. Biol. Chem. 49, 3077±3080. Yasumoto, T., Yotsu, M., Murata, M., Naoki, H., 1988. New tetrodotoxin analogues from the newt Cynops ensicauda. J. Am. Chem. Soc. 110, 2344±2345. Yasumoto, T., 1991. Puffer®sh toxin. In: Bureau of Environmental Health. Ministry of Health and Welfare, Japan (Ed.). The Manual for the Methods of Food Sanitation Tests. Food Hygenic Association, Tokyo, pp. 296±300. Yasumoto, T., Yotsu-Yamashita, M., 1996. Chemical and etiological studies on tetrodotoxin and its analogs. J. Toxicol. Toxin Reviews 15, 81±90. Yotsu, M., Iorizzi, M., Yasumoto, T., 1990. Distribution of tetrodotoxin, 6-epitetrodotoxin and 11-deoxytetrodotoxin in newts. Toxicon 28, 238±241. Yotsu, M., Hayashi, Y., Khora, S.S., Sato, S., Yasumoto, T., 1992. Isolation and structural assignment of 11-nortetrodotoxin-6(S)ol from the puffer Arothron nigropunctatus. Biosci. Biotech. Biochem. 56, 370±371. Yotsu-Yamashita, M., Schimmele, B., Yasumoto, T., 1999. Isolation and structural assignment of 5-deoxytetrodotoxin from the puffer ®sh Fugu poecilonotus. Biosci. Biotechnol. Biochem. 63, 961±963.