The Effects of Glutathione- S -Tranferase Polymorphisms on Sulforaphane Metabolism

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COLLEGE OF PUBLIC HEALTH AND HUMAN SCIENCES The Effects of Glutathione-S-Tranferase Polymorphisms on Sulforaphane Metabolism Jeannie Allen1,2,3, Anna Hsu1,4, PhD; Lauren Atwell1,4, MS, RD; John Clarke1,4, PhD; Deborah Bella1, PhD; Urszula Iwaniec1,4, PhD and Emily Ho1,4, PhD. 1School of Biological and Population Health Sciences, 2Howard Hughes Medical Institute Summer Undergraduate Research Program, Undergraduate Research Awards Program (URAP), 4Linus Pauling Institute, Oregon State University • Introduction • Cruciferous Vegetables and Cancer Prevention • Cruciferous vegetable intake has been shown to have an inverse association with cancer development. • One family of compounds that is suggested to have this effect is the isothiocyanates, which are found in the plant as their precursors, the glucosinolates. • Sulforphane (SFN) is an isothiocyanate whose precursor, glucoraphanin is found in high quantities in broccoli and broccoli sprouts. • Once SFN is produced in the body, it is further metabolized by the mercapturic acid pathway [Fig. 1] to produce SFN-Glutathione (SFN-GSH), SFN-Cysteinyl-Glycine (SFN-CG), SFN-Cysteine (SFN-Cys) and SFN-N-acetylcysteine (SFN-NAC). • Figure 1. Cellular metabolism of SFN in the Mercapturic Acid Pathway • Glutathione-S-Transferases (GSTs) • GSTs catalyze the first step of the mercapturic acid pathway by conjugating a glutathione group to SFN. • The glutathione-s-transferases are a family of enzymes that include 7 different isoforms. • GSTA1, GSTM1, GSTP1 and GSTT1 were examined. • SFN as well as metabolites of the pathway have been shown to have chemopreventive properties. • As much as 90% of the population is polymorphic in at least one GST and may derive different benefits from cruciferous vegetable consumption. • Study Design • Participants gave blood and urine samples after consuming broccoli sprouts • Genomic DNA was collected from whole blood for polymorphism analysis at baseline and SFN metabolite levels were determined by HPLC tandem mass spectrometry1. • GSTA1 and GSTP1 were analyzed by PCR-RFLP2,3,4 and GSTM1 and GSTT1 were analyzed by multiplex PCR3. Results (Continued) ) Figure 3. GSTM1 Null Showed Decreased Excretion of SFN-CG After Broccoli Sprouts Consumption. Table 3. Total SFN and metabolite levels in plasma between 3 and 6 hours after broccoli sprout consumption. Blood samples Urine Collection Results Table 1. Number of Polymorphisms in Study Participants. GSTM1 null individuals displayed lower levels of SFN-CG in the urine suggesting that they may metabolize SFN less efficiently. No differences were statistically different with other genotypes or other SFN metabolites. *represents data from one individual No significant differences were found between genotypes on total SFN and SFN metabolite levels in the urine or plasma. Thus, TOTAL SFN levels in blood or plasma were not affected by GST polymorphisms. Further analysis of specific metabolites ( SFN-GSH, SFN-CG, SFN-Cys and SFN-NAC) were performed. • Conclusions • GST Polymorphisms are highly prevalent in humans. • GSTA1, GSTM1, GSTP1 and GSTT1 polymorphisms had no effect on total levels of SFN and SFN metabolites in the plasma or urine. • GSTM1 null individuals showed decreased excretion of SFN-GSH and SFN-CG suggesting that they may metabolize SFN less efficiently. • Cohort studies suggest a weekly consumption of at least 5 servings of cruciferous vegetables to gain optimal chemo-preventative benefits of sulforaphane. • Individuals with GST polymorphisms may respond differently to cruciferous vegetables. Figure 2. GSTM1 Null Showed Decreased Excretion of SFN-GSH After Broccoli Sprouts Consumption. 97% of the subjects exhibited at least one polymorphism of the 5 sites that were examined. Most subjects showed more that one and the average number of polymorphisms was 2. Thus the prevalence of GST polymorphisms is high in a healthy adult population in Corvallis. Table 2. Total SFN and SFN metabolite levels in urine over 24 hours after broccoli sprout consumption. • Acknowledgements • Committee Members: • Dr. Emily Ho, Dr. Anna Hsu, Dr. Urszula Iwaniec • Ho Lab • Karin Hardin, Dr. Carmen Wong, Dr. Laura Beaver, Lauren Atwell, Greg Watson, Dr. John Clarke • Mass Spectrometry Facility • Dr. Fred Stevens and Jeff Morre • Dr. Kevin Ahern • Funding Sources • Howard Hughes Medical Institute Summer Fellowship (HHMI), OSU’s Environmental Health Sciences Center, Undergraduate Research Awards Program (URAP) and NCI/NIH • Hypothesis • Individuals with polymorphisms in GST genes may have altered metabolism and absorption of sulforaphane. • Aim 1: To determine the prevalence of GST polymorphisms in a healthy Corvallis population. • Aim 2: To further explore the differences in SFN metabolism with GSTA1, GSTP1, GSTT1 and GSTM1genotypes. • . GSTM1 null individuals displayed lower levels of SFN-GSH in the urine suggesting that they may metabolize SFN less efficiently. No differences were statistically different with other genotypes or other SFN metabolites. *represents data from one individual. • References • 1.Clarke, J.D., et al., Bioavailability and inter-conversion of sulforaphane and erucin in human subjects consuming broccoli sprouts or broccoli supplement in a cross-over study design. Pharmacol Res. 64(5): p. 456-63. • 2.Ping, J., et al., Genetic analysis of glutathione S-transferase A1 polymorphism in the Chinese population and the influence of genotype on enzymatic properties. Toxicol Sci, 2006. 89(2): p. 438-43 • 3. Oliveira, A.L., et al., GSTT1, GSTM1, and GSTP1 polymorphisms and chemotherapy response in locally advanced breast cancer. Genet Mol Res. 9(2): p. 1045-53 • 4. Lee, Y.L., et al., Glutathione S-transferase P1 gene polymorphism and air pollution as interactive risk factors for childhood asthma. Clin Exp Allergy, 2004. 34(11): p. 1707-13