Synthesis and characterization of aflatoxin B1 mercapturic acids and their identification in rat urine

Biologic effects of the hepatocarcinogenic mycotoxin aflatoxin B1 are principally induced by one of its metabolites, the exo-aflatoxin B1 epoxide which produces both DNA and protein adducts in vivo. Detoxication of the exo-aflatoxin B1 epoxide can be mediated in part by glutathione S-transferases whose induction could be important in chemoprotection interventions. Thus, biomarkers of the enzymatic conjugation of exo-aflatoxin B1 epoxide with glutathione may be important indices of protection against the toxic effects of this agent. Since glutathione conjugates undergo further metabolic processing in vivo to yield mercapturic acids, increased urinary excretion of exo-aflatoxin B1 mercapturate could be expected during chemoprotection intervention. To determine if this mercapturic acid could be used as a biomarker, techniques for its specific measurement were developed using monoclonal antibody immunoaffinity chromatography and reverse phase high-performance liquid chromatography with ultraviolet absorbance and mass spectral detection. First, a synthetic exo-aflatoxin B1 mercapturate was characterized using mass spectrometry, ultraviolet absorbance, circular dichroism spectrometry, and chemical derivatization. In vivo metabolite characterization was then facilitated by comparison with the synthetically prepared exo-aflatoxin B1 mercapturate and both aflatoxin B1-glutathione conjugate diastereoisomers. In rats, 1% of the aflatoxin dose was excreted as exo-aflatoxin B1 mercapturate within 24 h. The finding that exo-aflatoxin B1 mercapturate was excreted in urine in a dose-dependent manner provides the basis for investigating its applicability as a biomarker of glutathione S-transferase status in aflatoxin chemoprotection studies.