RESEARCH DESIGN AND METHODS

1. Metabolism And Activity Of Sulforaphane From Single Dose Of Broccoli Sprouts Tracy Beckmann1, John Clarke2, Anna Hsu1, Carmen Wong1,3, Karin Hardin1, Deborah Bella1, and Emily Ho1,3 1Department of Nutrition and Exercise Sciences, 2Molecular and Cellular Biology Program, and 3Linus Pauling Institute, Oregon State University, Corvallis, OR 97331 INTRODUCTION Many vegetables contain phytochemicals, naturally occurring chemicals, that may reduce the risk of chronic diseases such as cancer and heart disease. Studies have clearly shown that organosulfur compounds found in cruciferous vegetables such as broccoli demonstrate cancer preventative properties (1). These organosulfur compounds, or glucosinolates are bitter, sulfur-containing glucose precursors that are broken down in the body (Figure 1) into several different isothiocyanates via the enzyme myrosinase, present in both the plant cell and human gut microflora (2). Broccoli contains the glucosinolate glucoraphanin that when consumed is converted to the isothiocyanate sulforaphane. The anti-cancer effects of sulforaphane have been established in both cell and animal models (3). Although broccoli has a high capacity to produce sulforaphane, broccoli sprouts contain 10 to 100 times higher levels of sulforaphane than the mature heads, and consuming small amounts of these sprouts may provide beneficial effects just as effectively as larger amounts of the same variety of the mature vegetable (4). Further research in humans is being conducted to better understand how increasing dietary intakes of sulforaphane can slow or prevent cancer growth. The mechanisms behind sulforaphane’s anti-cancer activity are suggested to be related to the induction of anti-oxidant enzymes (Figure 1) and epigenetic alterations (5). Epigenetic alterations induce changes in gene expression without modifying the DNA sequence, thereby playing a role in gene regulation (6). In a diseased state such as cancer, tumor suppressor genes are “silenced.” Histone deacetylase (HDAC) deacetylates histones, preventing DNA polymerase access for transcription of specific genes, such as the tumor suppressor genes. This chain of events may lead to unregulated cell growth and tumor development (7). There is increasing evidence that dietary compounds such as sulforaphane may act as HDAC inhibitors resulting in re-activation of these silenced genes (6). To compare the differences in metabolism of sulforaphane due to whole food versus supplement intake, after a one-month washout period the exact protocol will be repeated in the same subjects with a broccoli extract supplement (or placebo) rather than fresh sprouts. Metabolites in the blood and urine samples and epigenetic markers in the blood samples will be analyzed. We hope to increase our knowledge of sulforaphane metabolism and to provide evidence supporting dietary broccoli sprout consumption as an effective means of reaching physiological levels of sulforaphane that may exert anti-cancer effects. Plasma and white blood cells were collected. These samples will be analyzed for sulforaphane metabolite levels, histone deacetylase activity, and gluthathione-s-transferase polymorphism identification. Complete urine samples were collected over each time period. Subjects were provided with labeled 3 L collection containers to collect the urine samples. All urine (none was discarded) was collected between 0 and 3 hour, 3 and 6 hour, 6 and 12 hour,12 and 24 hour, and 24 and 48 hour time points. Subjects brought urine specimens to lab when they came in for blood draw. The volume of urine was recorded and two 10 ml aliquots were frozen at -80 °C. Urine samples will be analyzed for sulforaphane as well as sulforaphane metabolites (SFN-Cys, SFN-NAC, and SFN-GSH). The quantitative sulforaphane and sulforaphane metabolite analysis of the plasma and urine will take place at the EHSC mass spectrometry facility on campus. RESULTS Broccoli Sprout Study Timeline Pre-study Meeting Subject Recruitment Phase I Phase II February March April May Figure 4. Subject Recruitment. Over 50 members of the greater Corvallis community were interested in participating, but only 16 were required for this study. Figure 5. Ethnicity of Subjects. Recruitment was not limited by race or ethnicity. Phase I and Phase II Schedule Table 3. Subject Demographics. Means and standard deviations of various demographic parameters of subjects participating in the study. Control group is (n=4) and broccoli sprout group (n=12). Table 2. Vegetables to Avoid. Subjects were asked to avoid vegetables containing organosulfur compounds for 1 week prior to the study and throughout study duration. Phase I: Broccoli Sprout vs. Alfalfa Sprout Phase II: Freeze-dried broccoli supplement vs. Placebo Day 1: Wed 4/07 Day 2: Thur 4/08 Day 3: Fri 4/09 Day 1: Wed 5/05 Day 2: Thur 5/06 Day 3: Fri 5/07 Date Time (hr) after ingestion 24 48 24 48 Blood (15mL) (10 mL) (10mL) Figure 2. Study Timeline. Healthy adult subjects aged 18- 60 yrs. were recruited and screened via phone interviews February-March, 2010. Participants and alternates were required to attend the Pre-study Meeting on March 31 to sign consent forms and complete a general health questionnaire. Subjects were instructed on dietary restrictions (Table 1) and methods for food recall and urine collection. Phase I of the study took place April 7-9, while Phase II will occur May 5-7. Urine Dietary Records (3day) Control Group (n=4) Brocolli Sprouts (n=12) • PROGRESS TO DATE • Phase I is completed. A total of 16 subjects were recruited and participated in Phase 1 of the study. To date, urine and blood collections have been processed for analysis of sulforaphane metabolites and epigenetic markers, and analysis of dietary records are underway. • Phase II begins May 5th. After a one-month wash out period, the study protocol will be repeated utilizing a freeze-dried broccoli sprout supplement (or placebo) rather than fresh broccoli sprouts. • Blood and urine samples will be analyzed for sulforaphane metabolites by mass spectrometry. • Epigenetic markers will be analyzed, including: • HDAC activity • Histone acetylation status • Expression of tumor suppressor genes including cyclin D, p21, Bax. Figure 1. Sulforaphane Metabolism. This process involves (A) hydrolysis of glucosinolate to its isothiocyanate via myrosinase enzyme activity and (B) metabolism of SFN via the mercapturic acid pathway. GST, glutathione-S-transferase; GTP, γ-glutamyltranspeptidase; CGase, cysteinylglycinase; HAT, histone acetyltransferase. 0 6 12 3 3 6 12 0 Tue 5/04 Tue 4/06 Figure 3. Daily Study Timeline. Daily requirements of study participants included blood and urine collection at 0, 3, 6, 12, 24, and 48 hours after sprouts consumption. Dietary records are kept for the day prior to sprouts consumption through Day 2. • HYPOTHESIS AND GOALS • Hypothesis: • Broccoli sprouts (as a whole food) will be absorbed and metabolized more easily by humans than the supplement form. • b. Dietary intake of sulforaphane will induce epigenetic changes in humans such as decreasing HDAC activity and increasing gene expression of specific tumor suppressor genes. • Long Term Goals: • a. To determine the specific pathways in which sulforaphane is metabolized in humans. • b. To determine other epigenetic effects caused by the consumption of dietary sulforaphane by humans. Wash out period ACKNOWLEDGEMENTS Funding provided by the National Institutes of Health; National Cancer Institute. Conflict of interest statement: None declared. • REFERENCES • Zhang, Y., et al. Proc. Natl Acad. Sci. USA. (1992). 89, 2399-2304. • Verkerk, R., et al. Molecular Nutrition & Food Research. (2009).53(S2), S219-S265 • Myzak, M., et al. Carcinogenesis. (2006). 27(4), 811-819. • Fahey, J., et al. Proceedings of the National Academy of Sciences of the United States of America. 94(19), 10367-10372. (1997). • Clarke, J., et al. Cancer Letters. (2008). 269(2), 291-304. • Dashwood, R. & Ho, E. Seminars in Cancer Biology (2007). 17, 363-369. • Ho, E., Clarke, J., & Dashwood, R. Journal of Nutrition (2009). 139(12), 2393-2396. RESEARCH DESIGN AND METHODS The goal of this study is to examine the absorption, metabolism, and activity of sulforaphane derived from either broccoli sprouts or a purified broccoli extract supplement in human volunteers. The cross-sectional study currently underway has recruited healthy (Table 1) adult subjects (n=16: experimental=12, control=4) aged 18- 60 years old. For the first phase of this study (completed in early April), subjects consumed 40 g of broccoli sprouts (alfalfa sprouts for control group), then provided blood and urine samples at 0, 3, 6, 12, 24, and 48 hours after broccoli sprout consumption. Subjects kept 3-day diet records to monitor dietary intake of vegetables , macronutrients and kcals. Table 1. Exclusion Criteria. Criteria factors were chosen based on the likelihood that they may alter the body’s metabolism. Day 1: At hour 0 (prior to broccoli sprout ingestion) baseline urine specimen was collected and15 ml of blood was drawn. Diet records from previous day were collected and reviewed. Subjects were then fed breakfast that included 40 g of broccoli sprouts (alfalfa sprouts for control group). They returned to the lab 3, 6, and 12 hours after broccoli sprouts consumption for urine and blood samples for the analysis of sulforaphane metabolites and epigenetic changes in blood. Days 2 and 3: At hour 24 and 48 (post-sprout ingestion),10 ml of blood was drawn and 12- and 24-hour urine specimens, respectively, were collected for the analysis of sulforaphane metabolites and epigenetic changes in blood. Diet records for study day 1 and 2 were collected and reviewed.