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Determining Estrogenicity of a Cytochrome P450-dependent metabolite of 3,3’-diindolylmethane (DIM)

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. What is Diindolylmethane?.

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Determining Estrogenicity of a Cytochrome P450-dependent metabolite of 3,3’-diindolylmethane (DIM)

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  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? • Acting as an anti-estrogen (Important for treatment of breast cancer) • Competes with endogenous estradiol • Produces a weaker estrogenic response • Acting as an estrogen • Promotes cell growth

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

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

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

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

  10. 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. Relevance

  11. Goals IDENTIFY POSSIBLE ESTROGENIC DIM METABOLITES: 3H-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)

  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 3H

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

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

  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.

  17. In vitro exposure to DIM using trout liver slices Juvenile Male (< 18 months) Sex determined and liver removed. 8 mm coring device Krumdieck Tissue Slicer 8 mm x 250 u m o Incubated at 14 C on orbital shaker (~90 RPM) in containers saturated with 95% O / 5% CO 2 2 refreshed at least every 12 hr. Enlargement of well 1 ml Hank’s media + 1% BSA, 0.1% gentamicin , with liver slice 25% fetal bovine serum, and test compound in DMSO (0.2% volume).

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

  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. Summary • 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.

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

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