FUTHER REPORT OF OCCURRENCE OF TETRODOTOXIN (TTX) AND NEW analogues IN THE ANURA FAMILY BRACHYCEPHALIDAE

PIRES Jr.,O.R.(1), MORALES, R.A.V.(1,2), A. SEBBEN(1), BLOCH Jr, C.(2), SCHWARTZ, C.A.(1)

(1)Laboratório de Toxinologia, Departamento de Ciências Fisiológicas, UnB, (2)Embrapa - Recursos Genéticos e Biotecnologia - DF

Tetrodotoxin (TTX) is a potent neurotoxin that occurs in a wide range of animals, among terrestrial organisms, TTX has been reported only in certain amphibians. In this work, the occurrence of TTX and analogues was examined in three braquicephalid species: Brachycephalus ephippium, B. nodoterga and B. pernix using LC-FLD and LC-MS/MS. The methanolic extracts were evaporated and ressuspended in deionized water. The semipurified extract was submitted to an HPLC system using a post-column fluorescent detection system (LC-FLD) and to an LC-ESI/MS/MS system, carried out under a selection ion mode (SIM). The retention time data from the LC-FLD system showed the presence of TTX, 4-epiTTX, 4,9-anidroTTX and TDA while the SIM data confirmed the presence of these compounds and revealed other analogs 11-norTTX-6(S)-ol, 5-deoxyTTX, 11-deoxyTTX, 11-oxoTTX, 6-epiTTX. Two new mass components were also identified by mass analysis, 348Da and 330Da. The identity of the 320Da, 302Da and 304Da ions was achieved by their fragmentation pattern and the formation of characteristic daughter ions such as TTX(302Da, 284Da, 256Da,178Da and 262Da), 4,9-anhydroTTX(256Da, 210Da, 195Da, 162Da and 135Da) and 5-deoxyTTX(286Da, 176Da, 162Da, and 146Da), according to Shoji, Y. et al., 2001(Analytical Biochem.290,10-17). The two unknown compounds showed daughter ions similar to TTX, suggesting new TTX analogues. The fragmentation of TTX and analogues prove to be a powerful tool to characterize this compound family. There are almost 4.000 species of amphibians in 26 recognized families, among these, only six were described containing TTX (Urodela: Salamandridae and Ambystomatidae, Anura: Bufonidae, Dendrobatidae, Brachycephalidae and Rhacophoridae), probable TTX are not limited to these few families of amphibians.

CORRESPONDENCE TO:

Pires Jr, O.R., Laboratório de Toxinologia, Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, 70910-900, DF, Brasil, Email: osmindo@unb.br