Detection and characterization of a glutathione conjugate of ochratoxin A.
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 microM OTA (1) in the presence of 5 mM GSH following incubation for 1 h with either horseradish peroxidase (HRP)/H(2)O(2), 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 approximately 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.
Dai
Jian
J
Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109-7486, USA.
Park
Gyungse
G
Wright
Marcus W
MW
Adams
Marissa
M
Akman
Steven A
SA
Manderville
Richard A
RA
D000818:Animals; D001711:Biotransformation; D004789:Enzyme Activation / Q000187:drug effects; D005296:Ferrous Compounds / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006873:Hydroquinones / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D009183:Mycotoxins / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009793:Ochratoxins / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010777:Photochemistry; D011809:Quinones / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet
D005296:Ferrous Compounds; D006873:Hydroquinones; D009183:Mycotoxins; D009793:Ochratoxins; D011809:Quinones; C025589:ochratoxin A; D006735:Horseradish Peroxidase; D005978:Glutathione; C031927:hydroquinone