Intestinal disposition of sulforaphane in rat ileum and its concentration dependent effects on gene expression of metabolizing enzymes and transporters.

B106

Introduction: Sulforaphane (SFN), an isothiocyanate derived from broccoli and cruciferous vegetables, has been identified as a potent chemopreventive agent because of its effect on enzymes involved in carcinogen elimination. In cultured cell lines and after treatment of animals with high doses, SFN has shown to be a potent inducer of enzyme systems such as glutathione S-transferases, glucoronyl transferases and quinone reductase. However, to optimize the prevention strategies using SFN, detailed intestinal disposition at various concentrations and its dose dependent effect on expression of enzymes has not been studied in detail. Methods: The intestinal disposition of SFN was studied at three different concentrations of 10, 50 and 100 μM by single-pass intestinal perfusion of rat ileum coupled with mesenteric vein sampling. Rat ileum segments (~10cm) were perfused with SFN solution in iso-osmolar ammonium acetate buffer. Samples were collected from the outlet perfusate and mesenteric vein at 5 min intervals over 60 min. SFN and major metabolites, the glutathione (SFN-GSH) and N-acetylcysteine conjugates, were analyzed by a specific LC-MS/MS method developed in our laboratory. Effect on mRNA expression of various enzymes and transporters in perfused ileum segment was quantitated by real-time PCR using SYBR green master mix. Results: SFN was well absorbed from the ileum as evidenced by the high disappearance rate from the perfused segment. The average P_{eff(perfusate)} value of SFN from the perfusate at 10μM was 2.73 x 10^-4 cm/sec. However, SFN appearance in mesenteric blood was very low (P_b(SFN) = 7.09 x 10^-6 cm/sec). It has been observed that in blood, SFN appears mainly as glutathione conjugate with P_b(SFN-GSH) as high as 1.96 x 10^-4 cm/sec. At increasing perfusate concentrations, the P_{eff(perfusate)} values of SFN remained constant, however, P_b(SFN) increased with a corresponding decrease in P_b(SFN-GSH). The mRNA of major intestinal metabolizing enzymes (glutathione-S-transferase isoforms, quinone reductase and metallothione) and an efflux membrane transporter (MRP-2) involved in cancer prevention were induced rapidly and substantially (> 2 fold) as a function of concentration within a limited perfusion time of 40-60 min. Conclusions: In conclusion, disposition of SFN and effect on gene expression is dependent on concentration. At higher concentrations, increased permeability of SFN with corresponding reduction in GSH permeability suggests some important implications in dose dependent cancer prevention properties of SFN because of altered pharmacokinetics. This detailed study of intestinal disposition of SFN may provide better insight into its activity as a cancer prevention agent and opportunities for designing prevention strategies optimized for SFN's pharmacokinetics. Acknowledgement: Financial support from NCI R03 CA105465-01