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  • Broccolinate

    1. 1. Identification & Isolation of the Chemoprotective Substances in Cruciferous Vegetables
    2. 3. “ If a cancerous ulcer appears upon the breasts, apply a crushed cabbage leaf and it will make it well.” Cato the Elder (Rome: 234-149 BC) [first recorded description of these plants as Brassica = cabbage]
    3. 5. “ I do not like broccoli. And I haven't liked it since I was a little kid and my mother made me eat it. I’m the president of the United States and I’m not going to eat any more broccoli.” George Bush 41st President of the U.S.
    4. 6. Consumption of 19 vegetables including sauerkraut, coleslaw, Brussels sprouts and broccoli Meta-analysis: Crucifer consumption and cancer risk Adapted from: Graham et al. (1978) JNCI 61:709-714 Kohlmeier & Su (1997) FASEB J. 11: A369 OR (Odds Ratio) servings/month grams/day
    5. 8. Cruciferae (Brassicaceae) Arabidopsis (thule cress) Brassica Eruca (Arugula) Raphanus (radish; daikon) Nasturtium (watercress) (incl. mustard greens, Canola, rutabaga, Chinese cabbage, collards, turnip) oleracea botrytis (cauliflower) capitata (cabbage) gemmifera (Brussels sprouts) italica (broccoli)
    6. 9. Glucosinolates <ul><li>Secondary aromatic metabolites found in 16 plant families </li></ul><ul><li>Over 121 glucosinolates (defined by side chains) identified in plant kingdom; 10 commonly found in brassica </li></ul>
    7. 10. Estimated Glucosinolate Intake Estimates of average per person daily intake of dietary glucosinolates range from 15-18 mg in Canada & U.S 22 mg in the Netherlands 30-50 mg in the UK 112 mg in Japan 1 Milford & Evans (1991) Outlook on Agriculture 20:31-37. 2 Mullin & Sahasrabudhe (1978) Nutr. Rep. Intl. 18:273-279. 3 Wattenberg et al. (1986) In Diet, Nutrition & Cancer , Eds. Hayashi et al.
    8. 11. Glucosinolates in Food McNaughton, British Journal of Nutrition. 2003, Vol 90(3):687-697 <ul><li>Highest concentration in cress (389 mg/100 gm) </li></ul><ul><li>Lowest in Chinese cabbage </li></ul><ul><li>Approximately 36% lost through cooking, more if microwaved </li></ul><ul><li>Destruction of flavonoids by </li></ul><ul><ul><li>microwaving: 97% </li></ul></ul><ul><ul><li>boiling: 66% </li></ul></ul><ul><ul><li>steaming: minimal loss </li></ul></ul>
    9. 12. Glucosinolates <ul><li>Dietary sources not absorbed by humans (as is true for many glycosides) </li></ul><ul><li>Crushing of cell walls or attack by pathogens liberates myrosinase enzymes (thioglucoside glucohydrolases) from compartmentalized myrosin cells </li></ul><ul><li>Myrosinase is also produced by gut flora </li></ul><ul><li>This hydrolyzes glucosinolates into glucose & unstable intermediates, that degrade into several metabolically active compounds: isothiocyanates, thiocyanates, nitriles </li></ul>
    10. 13. Isothiocyanates <ul><li>Sharp taste (“bitter mustard oils”) </li></ul><ul><li>Absorbed in the small intestine and colon </li></ul><ul><li>Metabolites found in urine 2-3 hours after ingestion of brassica vegetables (5-6 hours when vegetables are cooked) </li></ul><ul><li>ITC yield from glucosinolates in food is highly variable depending on </li></ul><ul><ul><li>plant source, cultivation technique, season </li></ul></ul><ul><ul><li>mode of preparation </li></ul></ul><ul><ul><li>gut pH </li></ul></ul><ul><ul><li>gut flora </li></ul></ul>
    11. 14. Isothiocyanate Activity <ul><li>Specific activity varies depending on predominant compounds released </li></ul><ul><li>Modify activity of biotransformation enzymes </li></ul><ul><ul><li>Variable effects on Phase I enzymes (specific to compound) </li></ul></ul><ul><ul><li>Induction of many Phase II enzymes </li></ul></ul><ul><ul><ul><li>Individual response varies depending on genetic polymorphisms </li></ul></ul></ul><ul><li>Net effect is increase in metabolism & detoxification of chemical carcinogens </li></ul><ul><ul><li>“ indirect antioxidants” </li></ul></ul><ul><ul><li>Prevention of damage to DNA </li></ul></ul>
    12. 15. Phase 2 Response from Fruits/Vegetables <ul><li>Watercress </li></ul><ul><li>Broccoli </li></ul><ul><li>Cauliflower </li></ul><ul><li>Cabbage </li></ul><ul><li>Brussels Sprouts </li></ul><ul><li>Arugula </li></ul><ul><li>Kale </li></ul><ul><li>Kohlrabi </li></ul><ul><li>Daikon </li></ul><ul><li>Red Radish </li></ul><ul><li>Horseradish </li></ul><ul><li>Chinese Cabbage </li></ul><ul><li>Bok Choy </li></ul>Spinach Beets Lettuce Chicory Mizuna Corn Peas Carrots Potato Tomato Apples Oranges Lemons Grapefruit Pears Rhubarb Mango Avocado Peach Nectarine Grapes Dates Figs Apricot Collards Turnips Leeks Garlic Chives Onion Blueberries Raspberries Plum Pumpkin Squash Green Beans Watermelon Canteloupe Honeydew Strawberries Oregano Basil Cardamon Cinnamon Cloves Pepper Noni Juice
    13. 16. ITCs with known biological activity <ul><ul><li>Sulforaphane </li></ul></ul><ul><ul><ul><li>Derived from glucoraphanin ( 4-methyl-sulfinylbutyl) </li></ul></ul></ul><ul><ul><ul><li>Methylthioalkyl side chain </li></ul></ul></ul><ul><ul><li>Phenylethylisothiocyanate (PEITC) </li></ul></ul><ul><ul><ul><li>Derived from gluconasturtiin ( 2-phenylethyl) </li></ul></ul></ul><ul><ul><ul><li>Aromatic side chain </li></ul></ul></ul><ul><ul><li>Allyl isothiocyanate (mustard oil) </li></ul></ul><ul><ul><ul><li>Derived from sinigrin (from black mustard) </li></ul></ul></ul><ul><ul><ul><li>Allyl side chain </li></ul></ul></ul><ul><ul><li>Indole-3-carbinol </li></ul></ul><ul><ul><ul><li>Derived from glucobrassicin ( 3-indolylmethyl) </li></ul></ul></ul><ul><ul><ul><li>Indole side chain </li></ul></ul></ul><ul><ul><li>3,3’-Diindolylmethane </li></ul></ul><ul><ul><ul><li>I3C condensation product </li></ul></ul></ul>
    14. 17. PEITC <ul><li>Watercress is a major source </li></ul><ul><li>Shown to inhibit chemically-induced carcinogenesis in rats </li></ul><ul><li>Promotes excretion of carcinogens </li></ul><ul><li>Induces apoptosis of human leukemia cells by activation of caspase pathway </li></ul>
    15. 18. PEITC Activity <ul><li>Inhibition of CYP 450 (Phase 1): 1A1, 1A2, 2B1, 2E1 </li></ul><ul><li>Induction of Phase II: </li></ul><ul><ul><li>Glutathione S-transferases </li></ul></ul><ul><ul><li>Quinone reductase </li></ul></ul><ul><ul><li>Epoxide hydrolase </li></ul></ul><ul><ul><li>UDP glucuronosyl transferase </li></ul></ul>
    16. 19. Indole-3-carbinol <ul><li>Product of myrosinase hydrolysis of glucobrassicin </li></ul><ul><li>Further metabolized (condensed) in acidic pH of stomach to complex mixture of compounds, including: </li></ul><ul><ul><li>Diindolylmethane (DIM) (I3C dimer) </li></ul></ul><ul><ul><li>Indolocarbazole (ICZ) </li></ul></ul><ul><ul><li>Ascorbigen (ASB) </li></ul></ul><ul><li>Shown to increase hepatic production of 2-hydroxyestrone relative to 16 alpha-hydroxyestrone </li></ul><ul><li>Inhibits breast, cervical and endometrial cancers when given orally </li></ul><ul><li>Ameliorates systemic lupus erythematosis in mice </li></ul><ul><li>Inhibits herpes virus in vitro </li></ul>
    17. 20. Indole-3-carbinol <ul><li>Majority of beneficial systemic effects probably mediated through DIM </li></ul><ul><li>In cell cultures, I3C is almost completely converted to DIM within 24 hours (therefore cell culture studies of I3C are actually measuring a DIM effect) </li></ul><ul><li>Other metabolites have toxic effects: </li></ul><ul><ul><li>Indolocarbazole has similar structure to TCDD (dioxin) & may be tumor promoter </li></ul></ul><ul><ul><li>Ascorbigen induces 4-hydroxylation of estrogen to make carcinogenic 4-OH E-2 </li></ul></ul>
    18. 21. Modification of Carcinogen Metabolism by Indole-3-Carbinol (I3C) <ul><li>“ When administered before carcinogen exposure, I3C reduces both the incidence of neoplasia & the formation of covalent adducts of carcinogen with DNA” </li></ul><ul><li>“ When administered after carcinogen exposure, I3C increases neoplastic outcome and, thus, promotes carcinogenesis” </li></ul>Bjeldanes et al. (1991) Proc. Natl. Acad. Sci. USA 88:9543-9547.
    19. 22. Int J Oncol. 2005 Nov;27(5):1391-9. Indole-3-carbinol inhibits the growth of human colon carcinoma cells but enhances the tumor multiplicity and volume of azoxymethane-induced rat colon carcinogenesis. Suzui M , Inamine M , Kaneshiro T , Morioka T , Yoshimi N , Suzuki R , Kohno H , Tanaka T . Tumor Pathology Division, Faculty of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan. Indole-3-carbinol (I3C) is a naturally occurring phytochemical which exerts a broad range of biological activities. The purpose of this study was to examine the effects of I3C on colon carcinogenesis, cell proliferation, cell-cycle progression and apoptosis, and on the levels of expression of several cell-cycle control molecules. We used a long-term rat model by using azoxymethane (AOM) to induce tumors (adenomas and adenocarcinomas) in the colon. In the present study, we found that after AOM injection, the treatment of male F344 rats with 0.01 and 0.05% I3C caused a significant increase in the tumor multiplicity of adenocarcinomas by 2.2- (P<0.05 for 0.01% I3C) and 2.1-fold (P<0.0002 for 0.05% I3C) respectively, when compared to the control rats. In addition, the tumor multiplicity of adenoma plus adenocarcinoma and the volume of adenocarcinoma were also increased by 2.0- (P<0.00001) and 2.1-fold (P<0.05) respectively, compared to the control. I3C significantly increased the proliferating cell nuclear antigen labeling index (PCNA LI) (P<0.008) and decreased the apoptotic index (P<0.05) of the colon adenocarcinoma. In contrast, in HCT 116 and HT29 human colon carcinoma cells, I3C inhibited growth and induced G1-phase cell-cycle arrest and apoptosis. Furthermore, I3C caused approximately a 2- to 4-fold increase in the cellular levels of p27KIP1 and p21CIP1 mRNA. These results suggest that I3C inhibits the growth of human colon carcinoma cells, at least in part, by inducing p27KIP1 and p21CIP1-mediated G1 cell-cycle arrest but dietary I3C promotes AOM-induced rat colon carcinogenesis by inhibiting the apoptosis of colon tumors. Therefore, the present study may provide further evidence for the ambivalent modulatory activity of I3C and this information may be useful when including I3C in cancer chemoprevention and/or extensive clinical therapy trials. PMID: 16211236 [PubMed - in process] These results suggest that I3C inhibits the growth of human colon carcinoma cells, at least in part, by inducing p27KIP1 and p21CIP1-mediated G1 cell-cycle arrest but dietary I3C promotes AOM-induced rat colon carcinogenesis by inhibiting the apoptosis of colon tumors.
    20. 23. 3,3’-Diindolylmethane <ul><li>Promotes 2OH-E2 production by inducing CYP1A1 </li></ul><ul><li>Inhibits 4OH-E2 production by blocking CYP1B1 </li></ul><ul><li>Modulatory effect on aryl-hydrocarbon receptor (blocks effects of endogenous estrogens and xenoestrogens) </li></ul><ul><li>Alleviates cyclic mastalgia & dysmenorrhea </li></ul><ul><li>Inhibits estrogen induced proliferation of MCF-7 cells in vitro </li></ul><ul><li>Promotes apoptosis in breast, cervical, prostate & colon cancer cells in vitro </li></ul><ul><li>Cytostatic effects on endometrial cancer in vitro </li></ul><ul><li>Inhibits growth of breast cancer cells in vivo </li></ul><ul><ul><li>Downregulates cyclin-D-kinases </li></ul></ul><ul><ul><li>Inhibits angiogenesis </li></ul></ul><ul><li>Inhibitory effects on human papillomavirus </li></ul>
    21. 24. Biochem Biophys Res Commun. 2005 Nov 25;337(3):1019-25. 3,3'-Diindolylmethane, a cruciferous vegetable derived synthetic anti-proliferative compound in thyroid disease. Tadi K , Chang Y , Ashok BT , Chen Y , Moscatello A , Schaefer SD , Schantz SP , Policastro AJ , Geliebter J , Tiwari RK . Department of Microbiology and Immunology, New York Medical College, Valhalla, NY, USA. Considerable epidemiological evidence exists to link thyroid disease with differing patterns of dietary consumption, in particular, cruciferous vegetables. We have been studying the anti-thyroid cancer (TCa) activity of indole-3-carbinol (I3C) found in cruciferous vegetables and its acid catalyzed dimer, 3,3'-diindolylmethane (DIM). There are no studies as yet to elucidate the effect of these compounds on the altered proliferative patterns in goiter or thyroid neoplasia. In this study, we tested the anti-proliferative effects of I3C and DIM on four different thyroid cancer cell lines representative of papillary (B-CPAP and 8505-C) and follicular carcinoma of the thyroid (CGTH-W-1 and ML-1), and primary human goiter cells. Cell survival and IC50 values for I3C and DIM were calculated by the XTT assay and cell cycle distribution analysis was done by flow cytometry. DIM was found to be a better anti-proliferative agent than I3C in both papillary and follicular TCa resulting in a greater cytotoxic effect at a concentration over three fold lower than predicted by the molar ratio of DIM and I3C. The anti-proliferative activity of DIM in follicular TCa was mediated by a G1 arrest followed by induction of apoptosis. DIM also inhibited the growth of primary goiter cells by 70% compared to untreated controls. Contrary to traditional belief that cruciferous vegetables are &quot;goitrogenic&quot;, DIM has anti-proliferative effects in glandular thyroid proliferative disease. Our preclinical studies provide a strong rationale for the clinical exploration of DIM as an adjuvant to surgery in thyroid proliferative disease. PMID: 16219298 [PubMed - in process] DIM was found to be a better anti-proliferative agent than I3C in both papillary and follicular TCa resulting in a greater cytotoxic effect at a concentration over three fold lower than predicted by the molar ratio of DIM and I3C.
    22. 25. Sulforaphane <ul><li>Discovered in 1992 at Johns Hopkins </li></ul><ul><li>Over 400 scientific papers now published </li></ul><ul><li>Rich in broccoli, Brussels sprouts, & kale </li></ul><ul><li>Potent transcription inducers of phase II detoxification & antioxidant enzymes: </li></ul><ul><ul><li>GST transferases </li></ul></ul><ul><ul><li>Epoxide hydrolases </li></ul></ul><ul><ul><li>Quinone reductases </li></ul></ul><ul><ul><li>Glucuronosyl transferases </li></ul></ul>
    23. 27. Sulforaphane <ul><li>Bacteriocidal against H. pylori </li></ul><ul><li>May protect against carcinogen (benzopyrene) induced gastric cancer </li></ul><ul><li>May Lower blood pressure </li></ul><ul><li>May Lower cholesterol </li></ul><ul><li>Prevention of AMD </li></ul><ul><li>Induces apoptosis of breast and prostate cancer cells in vitro </li></ul>
    24. 28. “ We have found that sulforaphane…is a potent bacteriostatic agent against….strains of H. pylori irrespective of their resistance to conventional antibiotics….sulforaphane blocked benzo[a]pyrene-evoked fore-stomach tumors… this protection resulted from induction of phase II detoxication and antioxidant enzymes.” PNAS; May 2002, Vol 99, no.11
    25. 29. Dietary Intake of Sulforaphane-Rich Broccoli Sprouts Improves Gastritis in H.pylori-Infected Human Subjects Abstract #3442 Akinori Yanaka, University of Tsukuba, Japan. Presented at the American Association for Cancer Research Meeting, Baltimore, MD; November 2, 2005 Yanaka and collegues found that over 2 months a diet rich in SGS significantly reduced Helicobactor pylori infection among a group of 20 individuals (vs 20 w/o SGS in diet)…he concluded that a diet rich in sulforaphane glucosinolate may help protect against gastric cancer, and hypothesized that this is due to the activation of gastric mucosal antioxidant enzymes that can protect the cells from H.pylori-induced DNA damage.
    26. 30. Rats were fed SGS…had a significant decrease in oxidative stress in cardiovascular and kidney tissues and increase in GSH content…decreased oxidative stress correlated with better endothelial-dependent relaxation of the aorta and significantly lower blood pressure. We conclude that a diet containing phase 2 protein inducers may also reduce the risk of developing cardiovascular problems of hypertension and atherosclerosis PNAS, May 2004; vol 101, no.18
    27. 31. “ . . . SF [sulforaphane] …activates transcription of phase II genes, whose products provide chemically versatile, often catalytic, and prolonged “indirect” antioxidant protection. . . . Intensification of endogenous … phase II enzymes by SF treatment may be a useful strategy to prevent photooxidative stress-related retinal diseases such as age-related macular degeneration, retinitis pigmentosa, and photic maculopathy.” Talalay, P Gao, X; PNAS; July 2004, vol 101, no 28
    28. 32. This is the first study to provide direct evidence that broccoli sprouts can enhance the body’s detoxifying system to help prevent cancer. Although previous laboratory studies indicated this was true, this is the first time that a direct observation of this effect in humans was possible. (200 adults) The blinded, placebo-controlled study was conducted in Jiangsu Province near Shanghai, a rural area where the incidence of liver cancer is extremely high due to consumption of foods tainted with aflatoxin …an inverse association was observed for excretion of diithiocarabmates and aflatoxin-DNA adducts in individuals receiving broccoli sprout glucosinolates Cancer Epidemiology Biomarkers & Prevention , Nov. 2005
    29. 33. Data from: Fahey et al. (Mar. 17, 2005) advance e-publication Carcinogenesis Khachick et al. (1999) Antioxidant Food Supplements in Human Health Fahey & Stephenson (2002) J.Agric. Food Chem. Phase 2 Response Induction by a Variety of Phytochemicals Potency -- Amount Required for CD ( µM) SULFORAPHANE PINOSTROBIN ZEAXANTHIN QUERCETIN CURCUMIN β -CAROTENE α -CAROTENE LUTEIN LYCOPENE CHLOROPHYLLIN VITAMIN B-12 CHLOROPHYLL
    30. 34. Phase 2 Enzyme Induction Potency of Broccoli: Effect of Plant Age 3-day sprouts seeds Market-stage broccoli heads Harvest-ready seed Inducer Activity Million Units / gram Plant Age (Days) Glucoraphanin Concentration
    32. 36. Lungs and Sulforaphane
    33. 37. Cytokine reduction with Sulforaphane
    34. 39. Breast and Sulforaphane
    35. 42. Sulforaphane and Prostate
    36. 46. Sulforaphane and Skin
    37. 50. Sulforaphane and Neurological