Further report of the occurrence of tetrodotoxin in Atelopus species (family: Bufonidae)

Toxicon. Vol. 33. No. 2. pp. 24&249. 1995 Copyright 0 1995 El&r Science Ltd Printed in Great Britain. All rights reserved 0041~)101/95 99.50+0.00

0041-0101(94)00149-9

FURTHER REPORT OF THE OCCURRENCE OF TETRODOTOXIN IN ATELOPUS SPECIES (FAMILY: BUFONIDAE) DIETRICH

MEBS,’ MART YOTSU-YAMASHITA,’ STEFAN Li)TTERS’ and ANDREAS

TAKESHI YASUMOT0,2 SCHLUTER3

‘Zentrum der Rechtsmedizin, University of Frankfurt, D-60596 Frankfurt, F.R.G.; 2Faculty of Agriculture, Tohoku University, Tsutsumidori-Amamiya, Aoba-ku, Sendai 981, Japan; and 3Staatliches Museum f. Naturkunde, D-70191 Stuttgart, F.R.G. (Received

19 July 1994; accepted

15 September

1994)

D. Mebs, M. Yotsu-Yamashita, T. Yasumoto, S. Liitters and A. Schhiter. Further report of the occurrence of tetrodotoxin in At&pus species (family: Bufonidae). Toxicon 33, 246-249, 1995.-In alcoholic extracts from museum samples of the toads Atelopus subornatus and A. peruensis tetrodotoxin and its analogues, 4-epitetrodotoxin and 4,9-anhydrotetrodotoxin were detected by fluorometric HPLC analysis, extending the list of amphibians containing this toxin. However, the toxin was not detected in samples of Melanophryniscus stelzneri, Dendrophryniscus minutus and Oreophrynella sp.

Beside its occurrence in a wide range of marine organisms, tetrodotoxin (TTX) has been found in skin and body extracts of various newts, salamanders, frogs and toads (Daly et al., 1987; Yasumoto et al., 1988; Yotsu et al., 1990). Recent studies suggest that the number of TTX-containing amphibian species is increasing: TTX has been found in several toads of the genus Atefopus (Bufonidae) as well as in a frog of the Dendrobatidae family, Colosthetus inguinalis (Daly et al., 1994). We report the occurrence of TTX in two more Atelopus species. Ethanol (70%) was used for TTX analysis, where museum specimens of Arelopus, Brachycephalus, Melanophryniscus, Dendrophryniscus and Oreophrynella sp. had been kept for preservation (at room temperature, some for several years; they had not been fixed in formalin, but preserved instead directly in ethanol). The alcoholic extracts were concentrated in oacuo 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. One mouse unit (MU) is equivalent to 0.22 pg TTX. Samples were analysed after clean-up (for Atelopus subornatus) with activated charcoal and a cation exchange gel (Hitachi 301 lc) or after ultrafiltration (Millipore C3, CL filter), by HPLC on a Develosil ODS-5 column using post-column reaction with 4 N NaOH and fluorescent detection as 246

241

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I

A

C 1

1

I

3

;i

*ry IO

15 Min.

200

15 Min.

-30

b

I

1

0

15 Yin.

30

Fig. 1. HPLC of alcoholic extracts from Arelopus species. (A) Ate/opus subornatus (3 years in captivity); (B) the same after clean-up with a Hitachi 301 lc column;(C) Afelopusperuensis. Tetrodotoxin (l), Cepitetrodotoxin (2) and 4,9-anhydrotetrodotoxin (3). 40

.6 20

16

856 “6.,6

I

Fig. 2. Mass spectrum of tetrodotoxin from Atelopus subornarus extract (major peak in Fig. 1B). The ion at m/z 319.6 corresponds to MH+ of TTX. rox 33,*--E

248

Short Communications Table 1. Results of mouse bioassay and HPLC analysis in extracts from Arelopus species Total mouse units (MU) in extract

TTX (MU) by HPLC

Atelopus subornatus

(Cordilleras near Bogota, Colombia, 1990) 1 specimen 3 years in captivity I specimen, 14 months in captivity

80

66

20

11

20

4

Atelopus peruensis

(Charco, 3.700 m, Huari, Ancash, Peru, April, 1989) 13 specimens Atelopus spumarius

(Serra do Navio, Brazil, 1975) 6 specimens

not detectable

previously described (Yotsu and Yasumoto, 1989; Yotsu-Yamashita et al., 1992). TTX was also identified by electrospray ionization (ESI) mass spectrometry (Finigan Mat TSQ 700). Of the various alcoholic extracts from toad samples, toxicity was only found in those from A. subornatus and A. peruensis. In these samples HPLC revealed peaks, which correspond to TTX as the major component and, in the case of A. subornatus, to its analogues 4-epi’TTX and 4,9-anhydroTTX. By mass spectrometry an ion was detected corresponding to MH+ or TTX (m/z 320) in the purified extract of A. subornatus. This confirms previous findings that besides TTX its analogues 4-epi and 4,9-anhydroTTX are the only minor components occurring in Atelopus species (Yotsu-Yamashita et al., 1992; Daly et al., 1994). It is interesting to note that the toxin is still present in A. subornatus, which had lived for 3 years in captivity. TTX was not detected in A. spumarius, where it had been identified by Daly et al. (1994). This may be due to a local variation of toxin content or due to the fact that the museum sample was 19 years old and the toxic activity may have been lost during storage. TTX was not detected in Dendrophryniscus minutus (20 specimens, Panguana, Rio Pachitea, Peru, Ott 1985) or Melanophryniscus stelzneri (22 specimens, Lome de Tigre, Calamuchita, Argentina, Jan 1988), confirming the findings of Daly et al. (1994). But TTX was also not present in extracts of the toad Brachycephalus ephippium (two specimens, Petropolis, Distr. Rio de Janeiro, Brazil, Jan 1983), where a TTX-like substance named ephippiotoxin has been reported (Sebben et al., 1986). Moreover, toads of the genus Oreophrynella (family: Bufonidae), which originate from very isolated areas, the tepuis in South Venezuela (one to three specimens each: Tranmen, Kukenan, Yuruani Tepui; Feb 1991), are essentially free of TTX. Although aqueous skin extracts of freshly collected amphibians might be most appropriate for TTX analysis, the present study confirms the previous experience that material from museum collections may represent a valuable source of information concerning the TTX distribution in terrestrial animals, e.g. amphibians, provided the material was not preserved in formalin, which rapidly destroys the toxin.

REFERENCES Daly, J. W., GaratTo, H. M. and Spande, T. F. (1993) Amphibian alkaloids. In: The Alkaloids, Vol. 43, pp. 185-288 (Cordell, G. A., Ed.). San Diego: Academic Press. Daly, J. W., Gusovsky, F., Myers, C. W., Yotsu-Yamashita, M. and Yasumoto, T. (1994) First occurrence of

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tetrodotoxin in a dendrobatid frog (Co/ostethus inguinalis), with further reports for the bufonid genus Atelopus. Toxicon 32, 279-285. Sebben, A., Schwartz, C. A., Valente, D. and Garcia Mendes, E. (1986) A tetrodotoxin-like substance found in the Brazilian frog Brachycephalus ephippium. Toxicon 24, 799-806. Yasumoto, T., Yotsu, M., Murata, M. and Naoki, H. (1988) New tetrodotoxin analogues from the newt Cynopsis ensicauda. J. Am. them. Sot. 110, 2344-2345. Yotsu, M. and Yasumoto, T. (1989) An improved tetrodotoxin analyzer. Agric. Biol. Cbem. 53, 893-895. Yotsu, M., Iorizzi, M. and Yasumoto, T. (1990) Distribution of tetrodotoxin, 6-epitetrodotoxin, and 1Ideoxytetrodotoxin in newts. Toxicon 28, 238-241. Yotsu-Yamashita, M., Mebs, D. and Yasumoto, T. (1992) Tetrodotoxin and its analogues in extracts from the toad Atelopus oxyrhynchus (family: Bufonidae). Toxicon 30, 1489-1492.