3.  Cancer is the second leading cause of death in the US [Centers for Disease Control and Prevention]. One in two men and one in three women will get some form of cancer in their lifetimes [American Cancer Society].  The total cost of cancer in 2010 was over 124 billion dollars and is projected to be 158 billion in 2020 [National Cancer Institute].

4.  Epidemiological studies have shown that an increase in cruciferous vegetable intake is correlated with reduced cancer risk [Clarke et al].  Isothiocyanates are one of the major anti- cancer bioactive compounds found in cruciferous vegetables, such as broccoli.

5.  Sulforaphane is an isothiocyanate found in cruciferous vegetables such as broccoli, broccoli sprouts, cauliflower and Brussels sprouts.  Sulforaphane has been shown to be an effective chemoprotective agent in vitro and in vivo by: › selectively inducing apoptosis in cancer cells › slowing tumor growth › Inhibiting HDAC activity [Ho et al] › Regulating phase I and II enzymes [Clarke et al]  Metabolism of sulforaphane in humans is not well known.

6.  Isothiocyanates are metabolized from their precursors, the glucosinolates. [Figure: Clarke, et.al.]  Myrosinase is found in cruciferous plants in the cell wall or in the human gastrointestinal microflora. ( Glucoraphanin )( Sulforaphane )

7.  The glutathione-s-transferase (GST) genes encode key enzymes (the GSTs) that are involved in the metabolism of sulforaphane.  SFN = Sulforaphane  SFN-GSH = Sulforaphane Glutathione  SFN-CG = Sulforaphane Cysteinylglycine  SFN-Cys = Sulforaphane Cysteine  SFN-NAC = Sulforaphane-N-Acetylcysteine  Polymorphisms are prevalent among humans with 90% or more of the population being polymorphic in at least one site [Ginsberg et al].

8.  Glutathione-S-transferases are a superfamily of enzymes that include 7 different isoforms.  The forms of the GST gene that were studied are GSTA1 (α), GSTP1 (π), GSTM1 (μ) and GSTT1 (θ) [ Prevalence distribution from Di Pietro et al, Ginsberg et al and Steck et al]. CaucasiansAsians African Americans Mexican Americans GSTM1 positive46%42-55%79%59% GSTT1 positive80%42-55%78%89% GSTP1 105 Ile/Ile30-55%44-68%6-53% GSTP1 105 Ile/Val34-65%25-50%39-80% GSTP1 105 Val/Val3-14%2-8%8-23% GSTP1 114 Ala/Ala82%95% GSTP1 114 Ala/Val18%5% GSTP1 114 Val/Val0% GSTA168% of the general population is polymorphic

9. To find out whether differences in an individual’s genotype in GSTM1, GSTT1, GSTP1 and GSTA1 affect their metabolism and excretion of sulforaphane. Individuals with polymorphisms of some of the GST genes may have altered metabolism and absorption of sulforaphane.

10.  Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)  GSTP1 is polymorphic at two sites: codon 105 and 114  Different polymorphisms are associated with higher or lower activity in a substrate dependent manner. Codon 105Codon 114 GSTP*A (wild type) Isoleucine (Ile)Alanine (Ala) GSTP*BValine (Val)Alanine GSTP*CValine GSTP*DIsoleucineValine

11. 3’5’ GSTP gene template Primer-forward Primer-reverse 3’ 5’ BsmA1 or Aci1 digestion of PCR product NNNNNNNNNNNNNGTCTCNNNNNNNNNNNNNNNNN BsmA1 DNA polymerase

13. 200 base pairs 100 base pairs  A single band at 176 base pairs indicates homozygosity (GSTP 105 Ile/Ile).  A band at 176 bp, two at 91 and 86 bp indicates that the individual is heterozygous (GSTP 105 Ile/Val).  Two bands at 91 and 86 bp without one at 176 bp indicates variant homozygosity (GSTP 105 Val/Val)  GSTA1 was analyzed in a similar fashion.

14.  GSTM1 and GSTT1 were analyzed using multiplex PCR.  For either gene, a person either expresses the gene (positive) or doesn’t (null). GSTT 480 bp Albumin 350 bp GSTM 215 bp

15. Genomic DNA was collected from whole blood for polymorphism analysis at baseline and SFN metabolite levels were determined by mass spectrometry. Participants gave blood and urine samples after consuming broccoli and alfalfa sprouts Broccoli sprouts (n=12) alfalfa sprouts (n=4)

16. PositiveNullHomozygous wtHeterozygousHomozygous variant GSTA1 --4 (25%)7 (43.75%)5 (31.25%) GSTP1 (105) --11 (68.75%) Ile/Ile 5 (31.25%) Ile/Val 0 Val/Val GSTP1 (114) --15 (93.75%) Ala/Ala 1 (6.25%) Ala/Val 0 Val/Val GSTM1 6 (37.5%) 10 (62.5%) --- GSTT1 11 (68.75%) 5 (31.25%) --- Distribution of GST Polymorphisms Among Study Subjects

17. Genotype Total amount of SFN metabolites excreted, µmols Homozygous Wild Type Heterozygous Homozygous Variant GSTA1 168.5 ± 90.2151.4 ± 21.1149.6 ± 35.6 GSTP1 (105) 171.6 ± 61.9 Ile/Ile 135.1 ± 33.1 Ile/Val N/A Val/Val GSTP1 (114) 162.7 ± 50.9 Ala/Ala 86.3 # Ala/Val N/A Val/Val GSTM1 179 ± 69.8140.2 ± 35 GSTT1 165.3 ± 56.4138.5 ± 36.1 # indicates n=1

18. Genotype Peak concentration of SFN metabolites in plasma, µmols/L Homozygous Wild Type Heterozygous Homozygous Variant GSTA1 2.4 ± 0.92.4 ± 0.22.3 ± 0.8 GSTP1 (105) 2.1 ± 0.5 Ile/Ile 2.7 ± 0.8 Ile/Val n/a Val/Val GSTP1 (114) 2.4 ± 0.7 Ala/Ala 1.4 # Ala/Val n/a Val/Val GSTM1 2.0 ± 0.62.5 ± 0.8 GSTT1 2.4 ± 0.62.3 ± 0.9 # indicates n=1

19. p-value Genotype0.0262 Time (hour)0.0001 Interaction0.1998

20. p-value Genotype0.0081 Time (hour)0.0003 Interaction0.1043

21.  The GSTA1, GSTP1, GSTM1 and GSTT1 polymorphisms that were examined have no effects on overall sulforaphane metabolism and excretion.  Individuals who are GSTM1 null excrete less SFN-GSH and SFN-CG, suggesting that they may metabolize sulforaphane less efficiently.  The National Cancer Institute recommends 5-9 servings of fruits and vegetables daily.  Cohort studies suggest a weekly consumption of at least 5 servings of cruciferous vegetables to gain optimal chemopreventative benefits of sulforaphane [Higdon].

22.  Dr. Emily Ho, Ph.D.  Dr. Anna Hsu, Ph.D.  Dr. John Clarke, Ph.D.  Ho lab › Karin Hardin › Carmen Wong, Ph.D. › Laura Beaver, Ph.D. › Lauren Atwell, M.S., R.D.  Mass Spectrometry Facility › Dr. Fred Stevens, Ph.D. › Jeff Morré  Dr. Kevin Ahern, Ph.D.  Funding sources: Howard Hughes Medical Institute Summer Fellowship (HHMI) and OSU’s Environmental Health Sciences Center (EHSC).

24. 1. Centers for Disease Control and Prevention. "Leading Causes of Death." FastStats. May 23, 2011. Centers for Disease Control and Prevention. August 16, 2011. http://www.cdc.gov/nchs/fastats/lcod.htm http://www.cdc.gov/nchs/fastats/lcod.htm 2. American Cancer Society. “Lifetime Risk of Developing of Dying From Cancer.” Learn About Cancer. August 10, 2011. American Cancer Society. August 16, 2011. http://www.cancer.org/Cancer/CancerBasics/lifetime-probability-of-developing-or- dying-from-cancer http://www.cancer.org/Cancer/CancerBasics/lifetime-probability-of-developing-or- dying-from-cancer 3. National Cancer Institute. The Cost of Cancer. [web] 2011 2/18/11 [cited 2011 August 16]; Available from: http://www.cancer.gov/aboutnci/servingpeople/cancer- statistics/costofcancer.http://www.cancer.gov/aboutnci/servingpeople/cancer- statistics/costofcancer 4. Clarke, J.D., R.H. Dashwood, and E. Ho, Multi-targeted prevention of cancer by sulforaphane. Cancer Lett, 2008. 269 (2): p. 291-304. 5. Ho, E., J.D. Clarke, and R.H. Dashwood, Dietary sulforaphane, a histone deacetylase inhibitor for cancer prevention. J Nutr, 2009. 139 (12): p. 2393-6. 6. Di Pietro, G., L.A. Magno, and F. Rios-Santos, Glutathione S-transferases: an overview in cancer research. Expert Opin Drug Metab Toxicol. 6 (2): p. 153-70 7. Ginsberg Gary, S.S., Hattis Dale, Guyton Kathryn Z., Johns Douglas O., and Babasaheb Sonawane, Genetic Polymorphism in Glutathione Transferases (GST): Population Distribution of GSTM1, T1, and P1 Conjugating Activity. Journal of Toxicology and Environmental Health, Part B, 2009. 12 : p. 389-439. 8. National Cancer Institute. Eat a Variety of Fruits & Vegetables Every Day. Accessed 9/22/08. Available at: http://www.fruitsandveggiesmatter.gov/.http://www.fruitsandveggiesmatter.gov/ 9. Higdon Jane, D.B., Williams David E., and Roderick H. Dashwood, Cruciferous vegetables and human cancer risk: epidemiologic evidence and mechanistic basis. Pharmacological Research, 2007. 55 : p. 224-236. 10. Steck, S.E., et al., GSTM1, GSTT1, GSTP1, and GSTA1 polymorphisms and urinary isothiocyanate metabolites following broccoli consumption in humans. J Nutr, 2007. 137(4): p. 904-9.