Detection and Characterization of a Glutathione Conjugate of Ochratoxin A

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Abstract
The ability of the carcinogenic mycotoxin ochratoxin A (OTA) to react with reduced glutathione (GSH) has been assessed using electrospray ionization (ES)-MS techniques. On the basis of the assumption that OTA undergoes biotransformation into the reactive quinone species OTQ (6), a synthetic sample of the reduced form of OTQ (6), hydroquinone OTHQ (5), was prepared and photoreacted with 6 M equiv of GSH to yield an authentic sample of the conjugate 8 that was definitively identified by mass spectrometry, UV−vis spectroscopy and NMR. With the authentic sample of 8 in hand, it was demonstrated that the same conjugate is produced from reaction of 100 μM OTA (1) in the presence of 5 mM GSH following incubation for 1 h with either horseradish peroxidase (HRP)/H2O2, rat liver microsomes (RLM)/NADPH or free Fe(II). In each of these oxidative systems the conjugate 8 was generated in less than 1% yield and the parent OTA molecule is poorly metabolized. Comparison of the peak area ratio of the conjugate 8 to that for the hydroxyOTA metabolite from the RLM/NADPH system implied that the conjugate was produced at a rate of ∼1−3 pmol min-1 (mg of protein)-1. These studies are the first to demonstrate that OTA undergoes biotransformation to a reactive intermediate [OTQ (6)] that covalently reacts with GSH to yield the conjugate 8. The biological implications of the reactivity of OTA toward GSH are discussed.

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