1. Determining Estrogenicity of a Cytochrome P450- dependent metabolite of 3,3’-diindolylmethane (DIM) Rachel O’Neal Susan Tilton Dr. David Williams Marine and Freshwater Biomedical Sciences Center Environmental and Molecular Toxicology Department

2. What is Diindolylmethane? Diindolylmethane (DIM) is the primary acid condensation product formed in the stomach after eating Indole-3-Carbinol Indole-3-Carbinol (I3C) is a compound naturally present in cruciferous vegetables

3. Indole-3-Carbinol Structure DIM I3C

4. DIM and I3C Currently promoted as chemopreventive agents (in Phase II clinical trials) Some studies suggest that I3C and DIM are chemoprotective in different target organs in various animal models. Other studies suggest that I3C and DIM can act as hepatic tumor promoters.

5. Potential Mechanisms of Tumor Modulation How can I3C and DIM prevent AND promote cancer? l Acting as an anti-estrogen (Important for treatment of breast cancer) n Competes with endogenous estradiol n Produces a weaker estrogenic response l Acting as an estrogen n Promotes cell growth

6. Potential Mechanisms of Tumor Modulation How can I3C and DIM prevent AND promote cancer? l Affects Metabolism of the Carcinogen n to increase excretion n to increase activation Carcinogen Reactive carcinogen metabolite Water soluble carcinogen metabolite Binds to DNA CANCERExcreted (P450) Phase I Phase II

7. Hypothesis DIM promotes liver tumors in trout through an estrogenic mechanism that is dependent upon conversion of DIM to estrogenic metabolites. P450 enzyme DIM DIM-OH OH Binds to estrogen receptor Mimics the effect of estrogen

8. Evidence for Estrogenic Metabolite P450 enzyme DIM DIM-OH OH Binds to estrogen receptor Mimics the effect of estrogen P450 Inhibition caused a decrease in the estrogenic response by DIM. Suggests DIM metabolite is an active estrogen.

9. Estrogenic Metabolite Evidence This data suggests that DIM metabolite(s) are the active estrogen(s).

10. Relevance Currently DIM is available over the counter at natural health food stores as a dietary supplement. DIM or DIM metabolites may exhibit potent estrogenic activity after metabolic activation and may play a role in tumor promotion in the liver.

11. IDENTIFY POSSIBLE ESTROGENIC DIM METABOLITES: 3 H-DIM metabolism High Performance Liquid Chromatography (HPLC) Mass Spectrometry (MS) COLLECT METABOLITE: Peak fraction collection (HPLC) DETERMINE METABOLITE ESTROGENICITY: Liver Slice experiment VTG induction (ELISA) Goals

12. Identifying possible estrogenic metabolites Incubated DIM with rat liver microsomes Compared samples incubated for zero minutes and 45 minutes for metabolite production 3H-DIM was used to increase sensitivity of metabolite detection DIM metabolites were analyzed by HPLC and LC/MS

13. 3H-DIM 3H

14. P450-dependent DIM Metabolism HPLC Chromatograms: 3 H Detection Time zero control 45 minute incubation DIM Metabolite

15. LC/MS (Mass Spectrometry) DIM-OH molecular weight = 262 DIM-OH

16. Examined estrogenicity of DIM metabolite Exposed trout liver slices to DIM metabolite Measured vitellogenin (VTG) protein in exposed liver slices by enzyme-linked immunosorbent assay (ELISA) Vitellogenin is an egg yolk protein synthesized in the liver of various egg-laying organisms after estrogen stimulation.

18. In vitro exposure to DIM using trout liver slices Estradiol DIM DIM met. Estradiol + DIM DMSO Estradiol + DIM Met.

19. Measure Vitellogenin (VTG) by ELISA Liver slices were homogenized in buffer and VTG induction was analyzed by ELISA. ELISA = Enzyme-linked immunosorbent assay VTG antibody competes for binding to purified VTG in plate or VTG in liver sample. VTG antibody is then detected by a substrate and changes color. Color change can be measured by absorbance on a spectrophotometer.

20. Estrogenicity of DIM Metabolite This data suggests that this metabolite of DIM is not estrogenic.

21. DIM’s estrogenicity can be inhibited by P450 inhibitors suggesting that DIM metabolite(s) have estrogenic activity. We have identified a metabolite that preliminary evidence by LC/MS suggests is a monohydroxylated form of the DIM that eludes from the HPLC at 19 minutes. We have determined that this metabolite is not estrogenic. Summary

22. Future Work Determine if metabolite binds to the estrogen receptor. Analyzing other DIM metabolites for estrogenicity. If we cannot find estrogenic metabolites, then determine other reasons P450 inhibition would reduce DIM estrogenicity.

23. Acknowledgements Dr. David Williams and lab Susan Tilton Marilyn Henderson Sharon Krueger Beth Siddens Yu Zhen EHSC Mass Spectrometry Facility Jeff Morre Marine/Freshwater Biomedical Sciences Center Environmental Health Sciences Center (EHSC) Howard Hughes Medical Institute