FIRST OCCURRENCE OF 11-OXOTETRODOTOXIN IN A TERRESTRIAL VERTEBRATE

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.

11-oxotetrodotoxin is a TTX analogue 3 to 4 four times more potent than TTX itself. It was discovery in the southern Pacific puffer fish Arothron nigropunctatus and, up to now, it was only described in two marine vertebrates. In this work we described the occurrence of this analogue in Brachycephalus ephippium from Atlantic rain forest using LC-FLD and LC-MS/MS systems. Eight adults specimens of B. ephippium were collected and brought alive to laboratory and killed by cooling up to a frozen state and submitted to toxin extraction with methanol 70%-acid acetic1%, evaporated and ressuspended in deionized water. The semipurified extract was submitted to a HPLC system using a post-column fluorescent detection system (LC-FLD) and to a LC-MS/MS system carried out in selection ions monitor mode (SIM) for m/z 320Da and 336Da corresponding to M+H⁺ of TTX and 11-oxoTTX respectively. These were chosen as precursor ions to be fragmented by collision-induced dissociation (CID).The comparison with retention time of synthetic 11-oxoTTX, produced by oxidation of TTX with Fenton’s reagent, in the LC-FLD system, and with SIM data confirm its identity. The CID data supports the identification of M+H⁺ 336Da as       11-oxoTTX showing some common guanidine ions of TTX family (162Da, 150Da and 178Da) and particular ions of 318Da, 300Da and 282Da corresponding to continuous loss of water from its structure. It is the first occurrence of 11-oxoTTX in an terrestrial vertebrate. The origin of this molecule in B. ephippium extracts is not yet understood but can be suggested that this formation occurs by transformation of some derivative present with TTX or even itself in this animal.

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