Human metabolites of brevetoxin PbTx-2: Identification and confirmation of structure

Abstract

Four metabolites were identified upon incubation of brevetoxin (PbTx-2) with human liver microsomes. Chemical transformation of PbTx-2 confirmed the structures of three known metabolites BTX-B5, PbTx-9 and 41, 43-dihydro-BTX-B5 and a previously unknown metabolite, 41, 43-dihydro-PbTx-2. These metabolites were also observed upon incubation of PbTx-2 with nine human recombinant cytochrome P450s (1A1, 1A2, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5). Cytochrome P450 3A4 produced oxidized metabolites while other CYPs generated the reduced products.

FIG. 1

Enzymatic and chemical conversion of PbTx-2. a) H₂/5% Pd-BaSO₄, THF, 0 °C, 32 psi, 2 h; b) NaBH₄, CH₃CN, rt, 0.5 h; c) NaClO₂, Na₂HPO₄, 2-methyl-2-butene, t-BuOH, RT, 4 h; d) NaClO₂, Na₂HPO₄, 2-methyl-2-butene; t-BuOH, RT, 4 h; e) NaBH₄, CeCl₃·6H₂O, MeOH, rt, 0.5 h.

FIG. 2

Total ion chromatogram (TIC) of metabolic products of 50 μM PbTx-2 incubated with pooled human liver microsomes (1 mg/mL) (a) 30 min. (b) 60 min. (c) 180 min. (d) Total ion chromatogram (TIC) of metabolic products of 50 μM PbTx-2 incubated for 30 min with 50 pmol CYPs.