Biotransformation of the naturally occurring isothiocyanate sulforaphane in the rat: identification of phase I metabolites and glutathione conjugates.
Sulforaphane (SFN) is a naturally occurring isothiocyanate present in cruciferous vegetables, such as broccoli, that has been identified as a potent inducer of glutathione S-transferase activities in laboratory animals. The present studies were carried out to elucidate the metabolic fate of SFN in the rat. Particular emphasis was placed on glutathione (GSH)-dependent pathways because conjugation with GSH is a major route by which many isothiocyanates are eliminated in mammals. Male Sprague-Dawley rats were administered a single dose of SFN (50 mg kg-1 ip), and bile and urine were collected over ascorbic acid. Analysis of biological fluids was carried out by ionspray LC-MS/MS using the neutral loss (129 Da) and precursor ion (m/z 164) scan modes to detect GSH and N-acetylcysteine (NAC) conjugates, respectively. In bile, five thiol conjugates (designated M1-M5) were detected. Metabolites M2 and M4 were identified as the GSH conjugates of SFN and erucin (ERN, the sulfide analog of SFN), respectively, by comparing their LC-MS/MS properties with those of standards obtained by synthesis. M1 was characterized as the GSH conjugate of a desaturated metabolite of SFN (tentatively assigned the structure of delta 1-SFN), suggesting that the parent compound also undergoes oxidative metabolism. Metabolites M3 and M5 were identified as the NAC conjugates of SFN and ERN, respectively, and together with the NAC conjugate of delta 1-SFN, these species also were detected in urine. Quantitative determination of the former two mercapturates in urine indicated that approximately 60% and approximately 12% of a single dose of SFN is eliminated in 24 h as the NAC conjugates of SFN and ERN, respectively. The corresponding figures in rats dosed with ERN were approximately 67% and approximately 29%. When the GSH conjugate of SFN was incubated with phosphate buffer (pH 7.4, 37 degrees C), < 1% of the conjugate dissociated to liberate free SFN. On the other hand, the conjugate underwent a facile thiol exchange reaction (> 70% conversion) when incubated in the presence of excess cysteine, thereby acting as an effective carbamoylating agent. It is concluded that SFN undergoes metabolism by S-oxide reduction and dehydrogenation and that GSH conjugation is the major pathway by which the parent compound and its phase I metabolites are eliminated in the rat.
Kassahun
K
K
Department of Medicinal Chemistry, University of Washington, Seattle 98195, USA.
Davis
M
M
Hu
P
P
Martin
B
B
Baillie
T
T
D000818:Animals; D001646:Bile / Q000737:chemistry; D001711:Biotransformation; D005978:Glutathione / Q000493:pharmacokinetics*; D005982:Glutathione Transferase / Q000378:metabolism; D017879:Isothiocyanates; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D013056:Spectrophotometry, Ultraviolet; D013454:Sulfoxides; D013861:Thiocyanates / Q000493:pharmacokinetics* / Q000652:urine
D017879:Isothiocyanates; D013454:Sulfoxides; D013861:Thiocyanates; D005982:Glutathione Transferase; C016766:sulforaphane; D005978:Glutathione