Determining Estrogenicity of a Cytochrome P450dependent 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?
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
Phase I
(P450)
Reactive carcinogen
metabolite
Phase II
Water soluble
carcinogen
metabolite
Binds to DNA
CANCER
Excreted
Hypothesis
DIM promotes liver tumors in trout through an
estrogenic mechanism that is dependent upon
conversion of DIM to estrogenic metabolites.

OH
N
DIM
N
H
H
N
N
P450
enzyme
H
DIM-OH
Binds to
estrogen
receptor
Mimics the
effect of
estrogen
H
Evidence for Estrogenic Metabolite
P450 Inhibition caused a decrease in the
estrogenic response by DIM.
Suggests DIM metabolite is an active
estrogen.


OH
N
DIM
N
H
H
N
N
P450
enzyme
H
DIM-OH
Binds to
estrogen
receptor
Mimics the
effect of
estrogen
H
VTG (ng/mg protein)
Estrogenic Metabolite Evidence
*
500
Controls
DIM or E2 + SKF 525A
DIM or E2 + Ketoconazole
250
0
E2
DIM
VTG induction in trout liver slices after 96 hour exposure to 100 nM E2 and 20 M DIM in the
presence or absence of 20 M P450 inhibitors .
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.
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)
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
3H
3H
N
N
H
H
P450-dependent DIM Metabolism
HPLC Chromatograms: 3H Detection
DIM
Metabolite
DIM
Time zero control
45 minute incubation
LC/MS (Mass Spectrometry)
DIM-OH
DIM-OH molecular weight = 262
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
Juvenile Male
(< 18 months)
Sex determined and liver removed.
8 mm coring device
KrumdieckTissue Slicer
8 mm x 250um
Incubated at 14 oC on orbital shaker (~90 RPM) in
containers saturated with 95% O 2/ 5% CO 2
refreshed at least every 12 hr.
Enlargement of well
with liver slice
1 ml Hank’s media + 1% BSA, 0.1% gentamicin,
25% fetal bovine serum, and test compound in
DMSO (0.2% volume).
In vitro exposure to DIM using trout liver slices
DMSO
Estradiol
DIM
DIM met.
Estradiol +
DIM
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
VTG (ng/mg)
2000
1000
20
10
0
DMSO
E2
DIM
Met
DIM+E2
Met+E2
This data suggests that this metabolite of DIM is not estrogenic.
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.
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