Determining Estrogenicity of a Cytochrome P450-dependent metabolite of

3,3’-diindolylmethane (DIM)

Rachel O’NealSusan Tilton

Dr. David Williams

Marine and Freshwater Biomedical Sciences Center

Environmental and Molecular Toxicology Department

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

Indole-3-Carbinol Structure

DIM

I3C

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.

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

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

Carcinogen Reactive carcinogen metabolite

Water soluble carcinogen metabolite

Binds to DNA

CANCER Excreted

(P450)

Phase IPhase II

Hypothesis

DIM promotes liver tumors in trout through an estrogenic mechanism that is dependent upon conversion of DIM to estrogenic metabolites.

P450 enzym

e

NHN

H

DIMNH

N

H

DIM-OH

OH Binds to estrogen receptor

Mimics the effect

of estrogen

Evidence for Estrogenic Metabolite

P450 enzyme

NHN

H

DIMNH

N

H

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.

Estrogenic Metabolite Evidence

E2 DIM0

250

500Controls

DIM or E2 + SKF 525A

DIM or E2 + Ketoconazole

VTG induction in trout liver slices after 96 hour exposure to 100 nM E2 and 20 μ M DIM in the 20presence or absence of μ 450 .M P inhibitors

*

VTG (ng/mg protein)

This data suggests that DIM metabolite(s) are the active estrogen(s).

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.

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 experimentVTG induction (ELISA)

Goals

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

3H-DIM

NHN

H

3H 3H

P450-dependent DIM Metabolism

HPLC Chromatograms: 3H Detection

Time zero control 45 minute incubation

DIM

DIM

Metabolite

LC/MS (Mass Spectrometry)

DIM-OH molecular weight = 262

DIM-OH

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.

In vitro exposure to DIM using trout liver slices

Enlargement of well with liver slice

Incubated at 14 oC on orbital shaker (~90 RPM) in containers saturated with 95% O 2/ 5% CO2refreshed at least every 12 hr.

1 ml Hank’s media + 1% BSA, 0.1% gentamicin, 25% fetal bovine serum, and test compound in DMSO (0.2% volume).

Juvenile Male(< 18 months)

Sex determined and liver removed.

8 mm coring device

KrumdieckTissue Slicer

8 mm x 250 um

In vitro exposure to DIM using trout liver slices

Estradiol

DIM

DIM met.

Estradiol + DIM

DMSO

Estradiol + DIM Met.

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.

Estrogenicity of DIM Metabolite

DMSO E2 DIM Met DIM+E2 Met+E20

10

20

1000

2000

VTG (ng/mg)

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

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

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.

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