1. Broccoli sprouts: An exceptionally rich source of inducers of enzymes that protect against chemical carcinogens Broccoli sprouts: An exceptionally rich source of inducers of enzymes that protect against chemical carcinogens Jed W.Fahey,Yuesheng Zhang, and Paul Talalay Brassica Chemoprotection lab-JHU Proc.Natl.Acad.Sci. USA,Vol.94.pp 10367-10372,Sept.1997 Medical Sciences

2. Review of Literature 1995 Talalay,P.- Toxicol.Lett. 82/83,173-179 metabolism chemoprotectors against cancer induce detoxication enzymes that take part in xenobiotic metabolism, and increase glutathione levels.

3. Literature review (cont.) Steinmetz,K.A&Potter,J.D-1996,Am.Diet.Assoc. Diets rich in fruits and vegetables are associated with decreased risk of developing cancer, partially due to the presence of phase 2 detoxication enzyme inducers.

4. Literature review (cont.) Prochaska,H.J.,Santamaria,A.B&Talalay-1988, Proc.Natl.Acad.Sci.USA 89,2394-2398 Crucifers of the Brassica genus (Broccoli,cauliflower,kale,brussles sprouts) contain large quantities of isothiocyanates (as glucosinolate precursors) specifically sulforaphane-a very potent inducer of phase 2 enzymes, and inhibitor of tumor formation in rats.

5. Literature review (cont.) Zhang,Y, Cho,C, Posner,G.H.&Talalay,P -1997 Anal.Biochem 205,100-107 Glucosinolates and isothiocyantaes are efficiently extracted from plants using a novel cold triple solvent method. Analysis is done by paired-ion chromatography and spectroscopy.

6. Hypothesis 3-day old broccoli sprouts contain extremely high concentrations of phase 2 enzyme inducers. These compounds are specific glucosinolates. The high potency extracts of these compounds are chemoprotectors against experimental mammary tumors.

7. Methodology Seeds not treated with pesticides obtained commercially, surface sterillized(with ethanol,bleach and detergent) and rinsed with distilled water. Sprouts are grown with 16-hr light and 8-hr darkness at 20 C in agar or at 25 C on trays, and collected immediately before extraction Mature and frozen vegetables obtained from store (stored at -80C before extraction).

8. Methodology(cont) Extraction of enzyme inducers Vegetables are homogenized with a triple solvent(dimethyl sulfoxide,dimethylformamide and acetonitrile) kept at -50C in dry ice/ethanol bath. Myrosinase enzyme is purified from daikon seedlings, used to hydrolyze glucosinolates for chemical and inducer activity assays,

9. Methodology(cont) Measurement of Isothiocynates and glucosinolates Spectroscopic analysis of Isothiocynate concentrations of plant extracts. Glucosinolates are converted to Isothiocynates by treatment with myrosinase and then analyzed by spectroscopy. Paired-ion chromatography was also used to analyze Glucosinolates

10. Methodology(cont) Inhibition of Mammary Tumor Development in rats Female rats were fed single 10-mg DMBA at age 50 days to initiate tumor formation. Glucosinolate or isothiocyanate preparations from broccoli sprouts and control were administered daily on days 47-51 (2 hr before the DMBA dose on day 50)

11. Methodology(cont) Plant preparation 3-day -old broccoli sprouts boiled in water for 3 min, cooled and made into powder. Powder dissolved in water, analyzed for inducer activity and for glucosinolates and isothiocyanate content. Excess myrosinase was added.

12. Results and Discussion The isothiocyanate sulforaphane is the principal and very potent enzyme inducer in broccoli. It is derived from glucoraphanin by the action of the myrosinase enzyme.

13. Results and Discussion(cont)

14. Results and Discussion(cont.) Fresh samples of broccoli had dramatically higher inducer activities than frozen samples. Both samples had no detectable activity before the addition of myrosinase, which proves that glucosinolates alone account for inducer activity. Blanching and unfavorable storage conditions account for removal of water soluble glucosinolates in the frozen samples and to reduced inducer activity.

15. Results and Discussion(cont.)

16. Inducer activity in broccoli sprouts was a 100 times greater than that in mature plants.

17. Results and Discussion(cont.)

18. Inducer potencies between sprouts and mature plants are associated with differences in the glucosinolates profile in these plants. Most glucosinolates in mature broccoli plants are indoles which are weak inducers of phase 2 enzymes, and potential tumor promoters.

19. Results and Discussion(cont.)

20. Mammary tumor development was significantly retarded compared with controls, and was dose-dependent for isothiocyanates, in DMBA-treated rats. Small amount of dried broccolli sprout extract (2.83% of daily diet) provided a highly significant tumor suppressor effect, greater then large amount of dried mature crucifers(40% of daily diet)

21. Conclusion Large quantities of inducers of enzymes that protect against carcinogens can be delivered in the diet by small quantities of crucifer sprouts, like 3 day old broccoli sprouts. High concentration of glucoraphanin, and low indole glucosinolates content is the key.

22. Future studies Since little is known about the metabolism of glucosinolates in humans, studies are underway that demonstrate efficient conversion of glucosinolates to isothiocyanates in humans in the absence of plant myrosinase.