TY - JOUR
T1 - Identification of 4-S-cysteinyltetrodotoxin from the liver of the puffer fish, Fugu pardalis, and formation of thiol adducts of tetrodotoxin from 4,9-anhydrotetrodotoxin
AU - Yotsu-Yamashita, Mari
AU - Goto, Ayako
AU - Nakagawa, Toshio
PY - 2005/5/1
Y1 - 2005/5/1
N2 - The metabolic pathway of tetrodotoxin (TTX), a powerful and specific voltage-gated sodium channel blocker, has not been well-clarified either in TTX-poisoned patients or in puffer fish. 4-S-CysteinylTTX (4-CysTTX) was isolated from the liver of the puffer fish, Fugu pardalis, as the first adduct of TTX with thiol. The structure was fully characterized by electrospray ionization-MS and two-dimensional NMR spectroscopy. The configuration of Cys in this compound was confirmed to be S (L-Cys) by application of the Marfey's method to cystine obtained from 4-CysTTX by iodine oxidation. We also found that 4-CysTTX was derived from 4,9-anhydroTTX by incubation with a large excess of Cys in aqueous buffer (pH 8.0) for 90 min at 40 °C in 33% yield by HPLC. GSH also reacted with 4,9-anhydroTTX to form 4-S-glutathionylTTX (4-GSTTX) in 39% yield under the same conditions, whereas TTX scarcely reacted with Cys and GSH. These reactions were strictly pH-dependent, giving the highest yield at pH 8.0. 4-GSTTX was converted to 4,9-anhydroTTX in 0.8 M sodium phosphate buffer (pH 8.0) at 25 °C. Its half-life was approximately 4 h. The minimum lethal doses of 4-CysTTX and 4-GSTTX to mice by ip injection were more than 140 and 860 μg/kg (n = 2), which were 14- and 86-fold larger than the LD50 of TTX, respectively. 4-GSTTX was hydrolyzed to 4-CysTTX by y- glutamyltranspeptidase (Sigma, catalog no. G9270), which was supposed to contain cysteinylglycine dipeptidase. We also examined the effect of Cys or GSH coinjection (ip) with TTX to mice for detoxification of TTX and concluded that these coinjections did not reduce the toxicity of TTX.
AB - The metabolic pathway of tetrodotoxin (TTX), a powerful and specific voltage-gated sodium channel blocker, has not been well-clarified either in TTX-poisoned patients or in puffer fish. 4-S-CysteinylTTX (4-CysTTX) was isolated from the liver of the puffer fish, Fugu pardalis, as the first adduct of TTX with thiol. The structure was fully characterized by electrospray ionization-MS and two-dimensional NMR spectroscopy. The configuration of Cys in this compound was confirmed to be S (L-Cys) by application of the Marfey's method to cystine obtained from 4-CysTTX by iodine oxidation. We also found that 4-CysTTX was derived from 4,9-anhydroTTX by incubation with a large excess of Cys in aqueous buffer (pH 8.0) for 90 min at 40 °C in 33% yield by HPLC. GSH also reacted with 4,9-anhydroTTX to form 4-S-glutathionylTTX (4-GSTTX) in 39% yield under the same conditions, whereas TTX scarcely reacted with Cys and GSH. These reactions were strictly pH-dependent, giving the highest yield at pH 8.0. 4-GSTTX was converted to 4,9-anhydroTTX in 0.8 M sodium phosphate buffer (pH 8.0) at 25 °C. Its half-life was approximately 4 h. The minimum lethal doses of 4-CysTTX and 4-GSTTX to mice by ip injection were more than 140 and 860 μg/kg (n = 2), which were 14- and 86-fold larger than the LD50 of TTX, respectively. 4-GSTTX was hydrolyzed to 4-CysTTX by y- glutamyltranspeptidase (Sigma, catalog no. G9270), which was supposed to contain cysteinylglycine dipeptidase. We also examined the effect of Cys or GSH coinjection (ip) with TTX to mice for detoxification of TTX and concluded that these coinjections did not reduce the toxicity of TTX.
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U2 - 10.1021/tx050015g
DO - 10.1021/tx050015g
M3 - Article
C2 - 15892580
AN - SCOPUS:18944398163
VL - 18
SP - 865
EP - 871
JO - Chemical Research in Toxicology
JF - Chemical Research in Toxicology
SN - 0893-228X
IS - 5
ER -