PHYTOESTROGENS/MYCOTOXINS MAY CAUSE ENDOMETRIOSIS

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710 K.M. Edmunds et al.

implants in women with endometriosis may

explain treatment failures and the persistence

of recalcitrant endometriosis in postmenopausal

women [6]. Therefore targeted

inhibition of local estrogen production in

endometriotic lesions by inhibition of aromatase

activity may have a place in the

management of this disease. As endometriosis

is the leading cause of hospitalization

for gynecologic surgery [10], thus novel,

safe and effective treatment options are

urgently needed.

Phytoestrogens are a class of plant estrogens

that include isoflavones, flavones, flavonones

and several mycotoxins such as

coumestrol and zeralanone. Phytoestrogens

are thought to have health benefits such as

providing protection against breast cancer

development [11, 12] and are potentially

useful in the management of menopausal

symptoms [13, 14]. Dietary factors such as

phytoestrogens have been shown to inhibit

aromatase activity [15, 16] without altering

plasma estrogen concentrations [17]. Therefore,

therapeutic use of phytoestrogens may

be of benefit to women with endometriosis.

Soy-based foods have high phytoestrogen

content of which genistein is the dominant

isoflavone [18]. Chrysin, a flavone

found in the plant Passiflora coerula and

naringenin, a flavonone found in citrus

fruits, have been shown to inhibit aromatase

activity in hepatocytes and placental microsomes

in vitro [19, 20]. However, the

effects of these compounds on aromatase

expression are unknown. Furthermore, aromatase

expression is regulated via different

promoter regions in a tissue specific manner

[21] and thus the effects of phytoestrogens

on endometrial aromatase expression

and activity are also unknown. Therefore,

the objective of this study was to screen several

phytoestrogens for their ability to

directly inhibit aromatase activity and to

determine the effect of dietary phytoestrogens

on aromatase expression and activity

in human endometrial stromal cells. Genistein

and daidzein, the dominant phytoestrogens

in the diet, together with chrysin and

naringenin, two phytoestrogens previously

shown to inhibit aromatase activity were

selected as the test compounds for this

study. We hypothesized that phytoestrogens

will inhibit aromatase activity in

endometrial stromal cell cultures and thus

potentially provide a novel therapeutic

option that is both natural and effective in

the management of endometriosis.

2. MATERIALS AND METHODS

2.1. Cell-free assay

The ability of the test compounds to

interact directly with the enzyme to alter

aromatase activity was investigated in a cell

free assay by modification of the fluorescence

assay described previously [22],

using human recombinant aromatase

expressed in insect cell microsomes (CYP19

suprasomes BD Gentest Biosciences,

Woburn, USA) and 0.25 µM dibenzylfuorescein

(BD Gentest Biosciences, Woburn,

USA) as the substrate. The ability of test

compounds (1 pM–100 µM in 0.1 M potassium

phosphate buffer pH 7.4) to inhibit

aromatase enzyme activity (0.4 pmol aromatase/

well was examined by incubation in

the presence of cofactors (40 µM NADP,

100 µM Glucose-6-phosphate, 100 µM

MgCl2) and DMSO (1%). Assays were performed

in a 96-well black walled culture

plate (Becton Dickinson, lin Lakes

USA) in a total volume of 202 µL. Reactions

were started by addition of 50 µL of

prewarmed (37 °C) enzyme to the prewarmed

plates. Blank wells contained

50 µL of buffer in place of the enzyme. The

plate was incubated at 37 ºC for 1 h and preliminary

experiments showed that enzyme

activity was linear up to 90 min. The reaction

was stopped by the addition of 75 µL

of 2 M NaOH to each well. Fluorescence

was measured using a PerkinElmer HTS

7000 Bio Assay Reader at an excitation

wavelength = 485 nm and emission = 535 nm.

Fluorescein (Sigma Aldrich, Oakville,

Canada) was used as the standard.

Genistein induced increased aromatase activity 711

Non-linear least-squares regression analysis

was used to fit inhibition curves to the

equation:

where Emax and Emin are the maximum and

minimum effects of the test compound,

respectively. pIC50 is the negative log of the

molar concentration of the compound that

produces 50% inhibition of enzyme and log

C is the molar concentration of the compound

that produces the effect E. IC50 values

were converted to Kis using the Cheng

Prusoff equation [23]:

Ki = IC50/(1 + S/Km))

where S is the substrate concentration and

Km is the is constant for the

enzyme. The Km and maximum velocity

(Vmax) of the enzyme reactions were determined

under the same conditions as

described for inhibition experiments except

that the substrate concentration varied

between 0 and 0.4 µM. Data for these experiments

were fit by non-linear least-squares

regression to:

V = (Vmax × S)/(Km + S)

where V is the reaction velocity at substrate

concentration S.

2.2. Endometrial stromal cell culture

Endometrial biopsies were obtained

from eighteen women aged 27–44 (mean

(± SD) of 38.3 ± 6.0 years) undergoing

benign gynecologic surgery at McMaster

University Medical Centre. Informed consent

was obtained from each patient by a

research nurse and all procedures were carried

out in accordance with approval of the

McMaster University Research Ethics

Board. Among the eighteen patients included

in this study, eleven had a laparoscopic

diagnosis of endometriosis and seven did

not have any evidence of pelvic endometriosis.

None of the study subjects had

received endocrine therapy in the previous

six months before surgery. Endometrial tissue

(1–2 g) obtained at hysterectomy was

rinsed in Hanks' balanced salt solution (HBSS)

containing 200 units·mL–1 penicillin,

0.2 mg·mL–1 streptomycin and 0.5 µg·mL–1

amphotericin B (Sigma Aldrich, Oakville

Canada) to remove blood and debris. Separation

of the endometrial stromal cells was

performed as previously described [24].

Briefly, the tissue was minced into 1 mm3

fragments and digested for 2.5 h at 37 °C

in medium containing collagenase type IA

(2 mg·mL–1, Sigma-Aldrich, Oakville,

Canada). After digestion, the remaining tissue

fragments were mechanically dispersed

and the dispersed cells were filtered

through a 100 µm and subsequently a

40 µm cell strainer (Becton Dickson, lin

Lakes, USA). Centrifugation (10 min, 725 ×

g) was used to pellet the cells after which

time they were resuspended in 3 mL of plating

media [DMEM:F12, 4% FBS, 1% ITS+

and 1% antibiotic antimycotic solution

(100 units·mL–1 penicillin, 0.1 mg·mL–1

streptomycin and 0.25 g·mL–1 amphotericin

B (Sigma Aldrich, Oakville, Canada)].

Red blood cells were removed by layering

the cell suspension over 3 mL of Ficoll-

Paque PLUS (Amersham Biosciences,

Uppsala, Sweden) in a sterile 15 mL polypropylene

tube. The solution was centrifuged

for 10 min at 400 × g. The media/

Ficoll interface layer containing the stromal

cells was plated into 48 well Falcon tissue

culture plates (Becton Dickson, lin

Lakes, USA) at a density of 200 000 cells/

well/0.5 mL. Media was changed after 48 h

and the cells were treated after 96 h in culture,

when the cells were near confluence.

Purity of the cell preparation was confirmed

by immunostaining for vimentin (mesenchymal

cell marker) and cytokeratin (epithelial

cell marker) as described below.

2.3. Cell treatment and aromatase

activity assay

Cells were washed twice in HBSS and

incubated for a minimum of 1 h in serum-free

DMEM-F12 containing 100 units·mL–1

penicillin, 0.1 mg·mL–1 streptomycin and

E = Emin +

(Emax – Emin)/ 1 10 –pIC50 – logC

( + )

712 K.M. Edmunds et al.

0.25 µg·mL–1 amphotericin B (Sigma Aldrich,

Oakville, Canada) prior to treatment for 24 h

with increasing log concentrations (10–9–

10–4 M) of genistein, daidzein, naringenin

or chrysin (Sigma Aldrich, Oakville, Canada)

diluted in serum free media. To examine

the role of estrogen receptor mediated

effects, the cells were also treated with genistein

in the presence of a non-selective

estrogen receptor antagonist (ICI 182,780;

Tocris, Ellisville, USA). After 24 h, the

treatment media was removed and replaced

with 500 µL of [1ß-3H]-androstenedione

[2.5 µCi·mL–1] (Perkin Elmer, Boston, USA)

in DMEM-F12 (containing 100 units·mL–1

penicillin, 0.1 mg·mL–1 streptomycin and

0.25 µg·mL–1 amphotericin for 4 h at

37 °C.

Aromatase activity was assayed using a

radiometric technique that quantifies the

incorporation of tritium from [1ß-3H]-

androstenedione into 3H-labeled water as

previously described [25]. Briefly, aromatase

activity was determined by transferring

300 µL of the incubation medium to

glass tubes, adding 300 µL of dextran

coated activated charcoal (250 mg·mL–1,

BD Biosciences, Oakville, Canada) to each

tube and incubating for 2 h at 4° C. The

samples were then centrifuged (15 min,

2500 × g) and the tritiated water content was

determined by counting the supernatant in

5 mL of scintillation fluid (Aqueous Counting

Scintillant, Amersham, England) in a

liquid scintillation counter. To control for

variation in the number of cells in each well,

the aromatase activity was normalized to

the cell protein content in each well as

determined by the Bradford method. Due to

variation in basal aromatase activity between

patients, normalized aromatase activity was

converted to a percentage of the control

level for each culture. The aromatase assay

is based on the release of tritiated water and

the specificity of the assay was determined

by co-incubation with 4-hydroxyandrostendione

an irreversible inhibitor of the catalytic

activity of aromatase [26] to block the

formation of tritiated water.

2.4. Immunocytochemistry

Cells were seeded into 8 well Lab-Tek

chamber slides (BD Biosciences, Oakville,

Canada) at a density of 200 000 cells/well/

0.5 mL. Media was changed after 48 h and

the cells were treated with genistein (10–6)

after 96 h in culture. After 24 h of treatment,

the cells were fixed in 10% neutral buffered

formalin, washed in PBS, and endogenous

peroxidase activity was quenched by incubating

the cells in 3% hydrogen peroxide (in

methanol) for 5 min. The cells were washed

in PBS, incubated with the primary antibodies

(Dako Diagnostics, Mississauga,

Canada) for cytokeratin (1:50), and vimentin

(1:50) for 1 h at room temperature and

immunostaining was identified using

EnVision (Dako Diagnostics, Mississauga,

Canada) with diaminobenzidine (Sigma-

Aldrich, Oakville, Canada) as the chromogen.

The cells were counterstained with

Carazzi hematoxylin. For negative controls,

the cells were incubated with non-immune

serum in place of the primary antibodies. To

stain for the presence of aromatase in the

genistein treated cultures and untreated

controls, immunohistochemistry was performed

on the chamberslides using a primary

monoclonal mouse antibody against

human aromatase (1:50 Serotec, Raleigh,

USA). Immunostaining was identified with

the avidin-biotin-peroxidase technique using

the Vectastain kit (Vector Laboratories,

Burlington, Canada) with diaminobenzadine

as the chromogen and Carazzi hematoxylin

as a counter stain.

2.5. Statistical analyses

Data were analyzed for equal variance

and normal distribution. An effect of treatment

on ESC aromatase activity was tested

using a one-way analysis of variance

(ANOVA) and differences between doses

were determined using the Tukey multiple

comparison method. A p value < 0.05 was

considered to be statistically significant for

all procedures used.

Genistein induced increased aromatase activity 713

3. RESULTS

3.1. Recombinant human aromatase

activity

Aromatase activity in the presence of

increasing substrate yielded a Km of 0.26 µM

(pKm = 6.6 ± 0.2) and a Vmax of 2.2 ± 1 pmol

fluorescein released per mol enzyme per

minute (Fig. 1). Naringenin (Ki = 0.3 µM)

and chrysin (Ki = 1 µM) were potent inhibitors

of recombinant human aromatase

whereas genistein and daidzein were weak

(Ki > 50 µM) inhibitors (Fig. 2).

3.2. Aromatase activity after

phytoestrogen treatment

of endometrial stromal cells

Immunocytochemical staining for cells

of mesenchymal origin and epithelial cells

illustrated that our cultures consisted of

Figure 1. is-Menten plot of the dibenzyfluorescein

deakylase activity of recombinant

human aromatase determined as described in

materials and methods. Points represent means

and standard errors of triplicates within a single

experiment. Km and Vmax values from this experiment

were 0.22 µM and 1.4 pmol/pmol/min,

respectively.

Figure 2. Human recombinant aromatase activity as indicated by fluorimetrically

quantified DBF

dealkylase after treatment with naringenin, chrysin, genistein and dadizein.

Each data point is the

mean (± SEM) from three separated experiments. Naringenin and chrysin were

effective inhibitors

of the enzyme with a Ki = 0.3 and 1.0 µM, respectively, while genistein and

daidzein were ineffective

as shown by a Ki > 50 µM.

714 K.M. Edmunds et al.

greater than 99% endometrial stromal cells

(data not shown).

Phytoestrogen treatment did not attenuate

aromatase activity in ESC from women

with endometriosis (n = 11) at any concentration

tested (Fig. 3). However, genistein

(10–9–10–6 M) treatment of ESC from

women without endometriosis (n = 7) resulted

in a significant increase in aromatase activity

(P < 0.05) to approximately 150% above

the activity observed in untreated ESC from

the same patient (Fig. 4), whereas daidzein,

naringenin and chrysin treatment had no

effect. Furthermore, the genistein induced

increase in aromatase activity was not attenuated

by co-treatment with the estrogen

receptor antagonist ICI 182,780 (P > 0.1,

Fig. 5).

3.3. Immunocytochemistry

Immunopositive aromatase staining was

evident as a diffuse brown cytoplasmic precipitate

(Fig. 6) that was absent in control

cultures where the primary antibody was

substituted with non-immune serum. Immunopositive

staining was focally present in

some but not all genistein (10–6 M) treated

ESC from eutopic endometrium of women

without endometriosis. Moreover, no immunoreactive

aromatase staining was visible in

the untreated ESC taken from the same

patient.

4. DISCUSSION

The objective of the current study was to

screen dietary phytoestrogens for their ability

to inhibit human recombinant aromatase

activity and to determine the effect of dietary

phytoestrogens on endometrial stromal

cell aromatase activity in culture. Although

naringenin and chrysin inhibited aromatase

in our cell-free assay, they were ineffective

in endometrial stromal cell cultures from

Figure 3. Aromatase activity was unchanged in genistein treated endometrial

stromal cell cultures

from women with endometriosis (n = 11). The control bar represents the aromatase

activity from

the vehicle treated cells from each of the patients and the data bars represent

the aromatase activity

of the cells following treatment with genistein represented as percent of

control. The control value

has arbitrarily been set to 100% and data are presented as the mean ± SEM.

Genistein induced increased aromatase activity 715

Figure 4. The effects of genistein treatment for 24 h on aromatase activity in

endometrial stromal

cells obtained from eutopic endometrium of women without endometriosis (n = 7).

The control bar

represents the aromatase activity from the untreated cells from each of the

patients and the data bars

represent the aromatase activity of the cells following treatment with genistein

represented as percent

of control. The results are the mean (± SEM) from seven different cultures.

Values with different

superscripts are significantly (P < 0.05) different.

Figure 5. The effects of 10–6 M genistein (GEN) alone and in combination with

10–6 M ICI 182 780

(ICI), and non-selective estrogen receptor antagonist, on aromatase activity

obtained from the eutopic

endometrium of women without endometriosis (n = 3). The results are the mean (±

SEM) from

three different cultures. Means identified with a different letter were

significantly different (P =

0.008).

716 K.M. Edmunds et al.

women with and without endometriosis and

thus are unlikely to have any potential therapeutic

benefit in the management of

endometriosis. In contrast, genistein, the

dominant isoflavone found in soy-based

foods, was inactive in the cell-free assay but

to our surprise increased aromatase activity

in endometrial stromal cells of women

without endometriosis. These data suggest

that the observed effects of genistein are not

mediated through direct effects of genistein

on enzyme activity but indirectly via

enhanced aromatase expression in endometrial

stromal cells or via intermediates on

aromatase activity. This point is supported

by evidence of immunocytochemical staining

for aromatase in genistein treated but

not untreated cells. In our study, the concentrations

of genistein that were used to

treat the human endometrial stromal cells

(1 nM to 10 mM) correspond to the serum

concentrations of both Asian and Caucasian

women who are consuming soy-based

foods [27–29] and thus are considered to be

physiologically relevant. Taken together,

our results suggest that while phytoestrogens

may have health benefits such as the

proposed protection against breast cancer

development [11, 12], genistein is unlikely

to have any therapeutic value in the management

of endometriosis and more importantly

may increase aromatase activity in

the endometrium and thus could be an

important factor in the pathobiology of this

enigmatic disease.

In the present study, aromatase was not

detected by immunohistochemistry in control

cultures of endometrial stromal cells

from women without endometriosis. In

addition, aromatase activity of vehicle

treated endometrial cells was at background

levels for the assay and thus supports the

view that aromatase is either absent or

inhibited in the endometrium from women

without endometriosis. Our findings are in

agreement with prior studies in which aromatase

cytochrome P450 has been reported

to be expressed in the endometrium of

women with endometriosis but is either

absent [4], or expressed at low levels in the

endometrium of women without endometriosis

[30]. Therefore, the patients in the

current study were grouped into two categories:

endometriotic and non-endometriotic.

None of the phytoestrogens tested

inhibited aromatase activity of the ESC

from women with endometriosis. However,

in the current study, genistein-treatment of

Figure 6. Immunocytochemical staining for aromatase in untreated cells (A) and

cells treated with

10–6 M genistein ( reveals positive staining in the treated cells (arrows).

Genistein induced increased aromatase activity 717

ESC from women without endometriosis

induced an increase in aromatase activity to

150% of the untreated controls similar to

the findings using adrenocortical carcinoma

cell lines treated with herbicides [31,

32]. Furthermore, our results are harmonious

with the previous finding that genistein

(30 µM) increased aromatase activity 3 fold

in the H295R human adrenocortical carcinoma

cell line [33]. Hence, genistein treatment-

induced changes in aromatase activity

could lead to increased local levels of estrogens

in the endometrium. However, the

functional significance of genistein induced

changes in aromatase activity is unknown.

A previous study has demonstrated that

genistein treatment increased cell proliferation

and was weakly estrogenic in

endometrial stromal cell and Ishikawa cell

cultures [34]. However, genistein treatment

antagonized the effects of estradiol in these

cultures suggesting that genistein is a competitive

antagonist of estradiol. Therefore,

a genistein induced increase in aromatase

activity and local estrogen production in the

endometrium could be relevant in hypoestrogenic

states such as menopause. While

genistein treatment was without effect on

the endometrium of macaque monkeys

with surgically induced menopause [35],

our proposal is supported by the observation

that endometrial hyperplasia was significantly

more prevalent in postmenopausal

women receiving soy tablets vs. a reference

group that received a placebo [36]. Moreover,

a recent study [37] has also shown that

high dose phytoestrogens can reverse the

antiestrogenic effects of clomiphene citrate

on the endometrium. Hence, we propose

that the effects of soy isoflavones, including

genistein on the endometrium is complex

and requires further study.

The mechanism through which genistein

treatment increased aromatase activity in

the endometrium remains unknown. Although

estradiol has been shown to increase aromatase

activity in ESC cultures [38], several

distinct lines of evidence lead us to

suggest that genistein is not acting through

an estrogen receptor mediated pathway in

our cultures to increase aromatase activity.

Genistein is a preferential estrogen receptor

(ER)-ß agonist and has been shown to have

estrogenic actions in a variety of tissues in

the rat [39, 40]. However, ER-a, not ER-ß

is the dominant ER sub-type expressed in

the endometrium [41]. Furthermore, it is

unlikely that genistein causes stimulation

of aromatase in the endometrium by acting

through a functional estrogen receptor pathway

because we have shown that the stimulation

of aromatase in ESC by genistein is

not attenuated by co-treatment of the cells

with ICI 182,780 which is a non-selective

estrogen receptor antagonist. We therefore

propose that it is unlikely that genistein

stimulates aromatase activity in endometrial

stromal cell cultures by acting directly

via the ER. Alternatively, we propose that

genistein can stimulate aromatase activity

in the endometrium through inhibition of

phosphodiesterase activity and result in

increased levels of cAMP. Support for this

proposal comes from evidence that genistein

inhibits cAMP-phosphodiesterase

activity in a variety of cell types [42–44]. In

addition, aromatase expression in the

endometrium is regulated through cAMPinduced

promoter II [45] and cAMP-treatment

has previously been shown to result in

a 26–60 fold increase in endometrial aromatase

activity [46].

Soy products are widely believed by the

public to provide health benefits. The Food

and Drug Administration has released an

approval for foods that contain at least

6.25 g of soy protein/serving to contain a

cardiovascular health claim (November 10,

1999; No. 279) and this has led to a plethora

of soy-based and fortified foods as well as

soy supplements to emerge on to the market

[47]. Phytoestrogens are efficiently absorbed

after ingestion and their bioavailability is

high enough to have biological effects [48].

Furthermore, contemporary studies reveal

that non-Asian women are ingesting increasing

amounts of phytoestrogens in their diet

as part of a trend towards a healthier lifestyle

[49, 50]. Despite potential health benefits

for women of some age groups, we speculate

718 K.M. Edmunds et al.

that genistein consumption by women of

reproductive age may have associated health

risks. Moreover, epidemiological evidence

demonstrates that Oriental women have a

higher incidence of endometriosis than

Caucasian women suggesting a link between

endometriosis and dietary phytoestrogens,

as Asian diets are high in soy isoflavones

[51, 52]. Hence, consumption of soy products

by women of reproductive age may not

be without consequence for endometrial

aromatase activity and potentially endometriosis.

In summary, the results of this study

demonstrate that dietary compounds, such

as genistein which is present in foods

including soy milk and tofu that the general

public views as healthy alternatives to traditional

foods in the North American diet,

can increase the local production of estrogen

in the ESC. Genistein-induced changes

in endometrial aromatase activity may have

detrimental effects which could lead to

increased risk for estrogen-dependent diseases

which involve the dysregulation of

aromatase such as endometriosis, adenomyosis

and uterine leiomyomas.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the technical

assistance of Louise Beecroft, RN

and Vasko (Bill) Georgievski.

This study was supported by funding from the

Canadian Network of Toxicology Centres

(WGF) and the Canadian Institutes of Health

Research (MOP 62681; WGF and ACH).

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720 K.M. Edmunds et al.

To access this journal online:

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[Guest guest]

this might be the key words here

inhibits cAMP-phosphodiesterase activity

dont have this sorted in my head yet but there looks to be something

here.

------------------------------

The fungus Mucor racemosus as a model of phenotypic

multidrug resistance in lower eukaryotes

page 203-204

They

interpreted this result to indicate that a volatile factor is needed to maintain

mold growth and removal of

this factor by increased flow rate of gas resulted in growth in the yeast

morphotype. This apparent

relationship of dimorphism to respiratory flux was further studied by Paznokas

and Sypherd, who noted

that the change in 3'-5'-cyclic adenosine monophosphate (cAMP) levels as a

result of growth conditions

influenced dimorphism [21]. In fact, addition of exogenous cAMP prevents growth

of the mold

morphotype [22]. cAMP phosphodiesterase activities were found to be relatively

constant in both the

mold and yeast morphotypes leading to the conclusion that adenylate cyclase

activity may be responsible

for changes in internal cAMP levels [22]. Rose, et al. demonstrated in 1999 that

Ras genes in M.

racemosus exhibit differential expression in mold versus yeast and that

exogenous addition of the cAMP

analog dibutyryl-cAMP caused a strong repression of RAS1 (but not RAS3)

concurrent with the

inhibition of growth of the mold morphotype [23]. It is likely that cAMP plays a

role in the signal

cascade leading to the dimorphic shift from one morphotype to the other. More

recent work in this area

has implicated pkaR, a cAMP-dependent protein kinase regulatory subunit, which

when overexpressed

favors growth in the mold morphotype [24, 25].

http://www.formatex.org/microbio/pdf/Pages201-212.pdf

Secondary Metabolism and Pathogenicity Abstracts

201. Fungal recognition of legume flavonoids: potential action through

inhibition of cAMP phosphodiesterase activity.

http://www.fgsc.net/asilo99/posterabs5.htm

>

> DURING CHILD BAREING YEARS!

> Article published by EDP Sciences and available at

http://www.edpsciences.org/rnd or http://dx.doi.org/10.1051/rnd:2005055

>

>

> 710 K.M. Edmunds et al.

> implants in women with endometriosis may

> explain treatment failures and the persistence

> of recalcitrant endometriosis in postmenopausal

> women [6]. Therefore targeted

> inhibition of local estrogen production in

> endometriotic lesions by inhibition of aromatase

> activity may have a place in the

> management of this disease. As endometriosis

> is the leading cause of hospitalization

> for gynecologic surgery [10], thus novel,

> safe and effective treatment options are

> urgently needed.

> Phytoestrogens are a class of plant estrogens

> that include isoflavones, flavones, flavonones

> and several mycotoxins such as

> coumestrol and zeralanone. Phytoestrogens

> are thought to have health benefits such as

> providing protection against breast cancer

> development [11, 12] and are potentially

> useful in the management of menopausal

> symptoms [13, 14]. Dietary factors such as

> phytoestrogens have been shown to inhibit

> aromatase activity [15, 16] without altering

> plasma estrogen concentrations [17]. Therefore,

> therapeutic use of phytoestrogens may

> be of benefit to women with endometriosis.

> Soy-based foods have high phytoestrogen

> content of which genistein is the dominant

> isoflavone [18]. Chrysin, a flavone

> found in the plant Passiflora coerula and

> naringenin, a flavonone found in citrus

> fruits, have been shown to inhibit aromatase

> activity in hepatocytes and placental microsomes

> in vitro [19, 20]. However, the

> effects of these compounds on aromatase

> expression are unknown. Furthermore, aromatase

> expression is regulated via different

> promoter regions in a tissue specific manner

> [21] and thus the effects of phytoestrogens

> on endometrial aromatase expression

> and activity are also unknown. Therefore,

> the objective of this study was to screen several

> phytoestrogens for their ability to

> directly inhibit aromatase activity and to

> determine the effect of dietary phytoestrogens

> on aromatase expression and activity

> in human endometrial stromal cells. Genistein

> and daidzein, the dominant phytoestrogens

> in the diet, together with chrysin and

> naringenin, two phytoestrogens previously

> shown to inhibit aromatase activity were

> selected as the test compounds for this

> study. We hypothesized that phytoestrogens

> will inhibit aromatase activity in

> endometrial stromal cell cultures and thus

> potentially provide a novel therapeutic

> option that is both natural and effective in

> the management of endometriosis.

> 2. MATERIALS AND METHODS

> 2.1. Cell-free assay

> The ability of the test compounds to

> interact directly with the enzyme to alter

> aromatase activity was investigated in a cell

> free assay by modification of the fluorescence

> assay described previously [22],

> using human recombinant aromatase

> expressed in insect cell microsomes (CYP19

> suprasomes BD Gentest Biosciences,

> Woburn, USA) and 0.25 µM dibenzylfuorescein

> (BD Gentest Biosciences, Woburn,

> USA) as the substrate. The ability of test

> compounds (1 pM–100 µM in 0.1 M potassium

> phosphate buffer pH 7.4) to inhibit

> aromatase enzyme activity (0.4 pmol aromatase/

> well was examined by incubation in

> the presence of cofactors (40 µM NADP,

> 100 µM Glucose-6-phosphate, 100 µM

> MgCl2) and DMSO (1%). Assays were performed

> in a 96-well black walled culture

> plate (Becton Dickinson, lin Lakes

> USA) in a total volume of 202 µL. Reactions

> were started by addition of 50 µL of

> prewarmed (37 °C) enzyme to the prewarmed

> plates. Blank wells contained

> 50 µL of buffer in place of the enzyme. The

> plate was incubated at 37 ºC for 1 h and preliminary

> experiments showed that enzyme

> activity was linear up to 90 min. The reaction

> was stopped by the addition of 75 µL

> of 2 M NaOH to each well. Fluorescence

> was measured using a PerkinElmer HTS

> 7000 Bio Assay Reader at an excitation

> wavelength = 485 nm and emission = 535 nm.

> Fluorescein (Sigma Aldrich, Oakville,

> Canada) was used as the standard.

>

> Genistein induced increased aromatase activity 711

> Non-linear least-squares regression analysis

> was used to fit inhibition curves to the

> equation:

> where Emax and Emin are the maximum and

> minimum effects of the test compound,

> respectively. pIC50 is the negative log of the

> molar concentration of the compound that

> produces 50% inhibition of enzyme and log

> C is the molar concentration of the compound

> that produces the effect E. IC50 values

> were converted to Kis using the Cheng

> Prusoff equation [23]:

> Ki = IC50/(1 + S/Km))

> where S is the substrate concentration and

> Km is the is constant for the

> enzyme. The Km and maximum velocity

> (Vmax) of the enzyme reactions were determined

> under the same conditions as

> described for inhibition experiments except

> that the substrate concentration varied

> between 0 and 0.4 µM. Data for these experiments

> were fit by non-linear least-squares

> regression to:

> V = (Vmax × S)/(Km + S)

> where V is the reaction velocity at substrate

> concentration S.

> 2.2. Endometrial stromal cell culture

> Endometrial biopsies were obtained

> from eighteen women aged 27–44 (mean

> (± SD) of 38.3 ± 6.0 years) undergoing

> benign gynecologic surgery at McMaster

> University Medical Centre. Informed consent

> was obtained from each patient by a

> research nurse and all procedures were carried

> out in accordance with approval of the

> McMaster University Research Ethics

> Board. Among the eighteen patients included

> in this study, eleven had a laparoscopic

> diagnosis of endometriosis and seven did

> not have any evidence of pelvic endometriosis.

> None of the study subjects had

> received endocrine therapy in the previous

> six months before surgery. Endometrial tissue

> (1–2 g) obtained at hysterectomy was

> rinsed in Hanks' balanced salt solution (HBSS)

> containing 200 units·mL–1 penicillin,

> 0.2 mg·mL–1 streptomycin and 0.5 µg·mL–1

> amphotericin B (Sigma Aldrich, Oakville

> Canada) to remove blood and debris. Separation

> of the endometrial stromal cells was

> performed as previously described [24].

> Briefly, the tissue was minced into 1 mm3

> fragments and digested for 2.5 h at 37 °C

> in medium containing collagenase type IA

> (2 mg·mL–1, Sigma-Aldrich, Oakville,

> Canada). After digestion, the remaining tissue

> fragments were mechanically dispersed

> and the dispersed cells were filtered

> through a 100 µm and subsequently a

> 40 µm cell strainer (Becton Dickson, lin

> Lakes, USA). Centrifugation (10 min, 725 ×

> g) was used to pellet the cells after which

> time they were resuspended in 3 mL of plating

> media [DMEM:F12, 4% FBS, 1% ITS+

> and 1% antibiotic antimycotic solution

> (100 units·mL–1 penicillin, 0.1 mg·mL–1

> streptomycin and 0.25 g·mL–1 amphotericin

> B (Sigma Aldrich, Oakville, Canada)].

> Red blood cells were removed by layering

> the cell suspension over 3 mL of Ficoll-

> Paque PLUS (Amersham Biosciences,

> Uppsala, Sweden) in a sterile 15 mL polypropylene

> tube. The solution was centrifuged

> for 10 min at 400 × g. The media/

> Ficoll interface layer containing the stromal

> cells was plated into 48 well Falcon tissue

> culture plates (Becton Dickson, lin

> Lakes, USA) at a density of 200 000 cells/

> well/0.5 mL. Media was changed after 48 h

> and the cells were treated after 96 h in culture,

> when the cells were near confluence.

> Purity of the cell preparation was confirmed

> by immunostaining for vimentin (mesenchymal

> cell marker) and cytokeratin (epithelial

> cell marker) as described below.

> 2.3. Cell treatment and aromatase

> activity assay

> Cells were washed twice in HBSS and

> incubated for a minimum of 1 h in serum-free

> DMEM-F12 containing 100 units·mL–1

> penicillin, 0.1 mg·mL–1 streptomycin and

> E = Emin +

> (Emax – Emin)/ 1 10 –pIC50 – logC

> ( + )

>

> 712 K.M. Edmunds et al.

> 0.25 µg·mL–1 amphotericin B (Sigma Aldrich,

> Oakville, Canada) prior to treatment for 24 h

> with increasing log concentrations (10–9–

> 10–4 M) of genistein, daidzein, naringenin

> or chrysin (Sigma Aldrich, Oakville, Canada)

> diluted in serum free media. To examine

> the role of estrogen receptor mediated

> effects, the cells were also treated with genistein

> in the presence of a non-selective

> estrogen receptor antagonist (ICI 182,780;

> Tocris, Ellisville, USA). After 24 h, the

> treatment media was removed and replaced

> with 500 µL of [1ß-3H]-androstenedione

> [2.5 µCi·mL–1] (Perkin Elmer, Boston, USA)

> in DMEM-F12 (containing 100 units·mL–1

> penicillin, 0.1 mg·mL–1 streptomycin and

> 0.25 µg·mL–1 amphotericin for 4 h at

> 37 °C.

> Aromatase activity was assayed using a

> radiometric technique that quantifies the

> incorporation of tritium from [1ß-3H]-

> androstenedione into 3H-labeled water as

> previously described [25]. Briefly, aromatase

> activity was determined by transferring

> 300 µL of the incubation medium to

> glass tubes, adding 300 µL of dextran

> coated activated charcoal (250 mg·mL–1,

> BD Biosciences, Oakville, Canada) to each

> tube and incubating for 2 h at 4° C. The

> samples were then centrifuged (15 min,

> 2500 × g) and the tritiated water content was

> determined by counting the supernatant in

> 5 mL of scintillation fluid (Aqueous Counting

> Scintillant, Amersham, England) in a

> liquid scintillation counter. To control for

> variation in the number of cells in each well,

> the aromatase activity was normalized to

> the cell protein content in each well as

> determined by the Bradford method. Due to

> variation in basal aromatase activity between

> patients, normalized aromatase activity was

> converted to a percentage of the control

> level for each culture. The aromatase assay

> is based on the release of tritiated water and

> the specificity of the assay was determined

> by co-incubation with 4-hydroxyandrostendione

> an irreversible inhibitor of the catalytic

> activity of aromatase [26] to block the

> formation of tritiated water.

> 2.4. Immunocytochemistry

> Cells were seeded into 8 well Lab-Tek

> chamber slides (BD Biosciences, Oakville,

> Canada) at a density of 200 000 cells/well/

> 0.5 mL. Media was changed after 48 h and

> the cells were treated with genistein (10–6)

> after 96 h in culture. After 24 h of treatment,

> the cells were fixed in 10% neutral buffered

> formalin, washed in PBS, and endogenous

> peroxidase activity was quenched by incubating

> the cells in 3% hydrogen peroxide (in

> methanol) for 5 min. The cells were washed

> in PBS, incubated with the primary antibodies

> (Dako Diagnostics, Mississauga,

> Canada) for cytokeratin (1:50), and vimentin

> (1:50) for 1 h at room temperature and

> immunostaining was identified using

> EnVision (Dako Diagnostics, Mississauga,

> Canada) with diaminobenzidine (Sigma-

> Aldrich, Oakville, Canada) as the chromogen.

> The cells were counterstained with

> Carazzi hematoxylin. For negative controls,

> the cells were incubated with non-immune

> serum in place of the primary antibodies. To

> stain for the presence of aromatase in the

> genistein treated cultures and untreated

> controls, immunohistochemistry was performed

> on the chamberslides using a primary

> monoclonal mouse antibody against

> human aromatase (1:50 Serotec, Raleigh,

> USA). Immunostaining was identified with

> the avidin-biotin-peroxidase technique using

> the Vectastain kit (Vector Laboratories,

> Burlington, Canada) with diaminobenzadine

> as the chromogen and Carazzi hematoxylin

> as a counter stain.

> 2.5. Statistical analyses

> Data were analyzed for equal variance

> and normal distribution. An effect of treatment

> on ESC aromatase activity was tested

> using a one-way analysis of variance

> (ANOVA) and differences between doses

> were determined using the Tukey multiple

> comparison method. A p value < 0.05 was

> considered to be statistically significant for

> all procedures used.

>

> Genistein induced increased aromatase activity 713

> 3. RESULTS

> 3.1. Recombinant human aromatase

> activity

> Aromatase activity in the presence of

> increasing substrate yielded a Km of 0.26 µM

> (pKm = 6.6 ± 0.2) and a Vmax of 2.2 ± 1 pmol

> fluorescein released per mol enzyme per

> minute (Fig. 1). Naringenin (Ki = 0.3 µM)

> and chrysin (Ki = 1 µM) were potent inhibitors

> of recombinant human aromatase

> whereas genistein and daidzein were weak

> (Ki > 50 µM) inhibitors (Fig. 2).

> 3.2. Aromatase activity after

> phytoestrogen treatment

> of endometrial stromal cells

> Immunocytochemical staining for cells

> of mesenchymal origin and epithelial cells

> illustrated that our cultures consisted of

> Figure 1. is-Menten plot of the dibenzyfluorescein

> deakylase activity of recombinant

> human aromatase determined as described in

> materials and methods. Points represent means

> and standard errors of triplicates within a single

> experiment. Km and Vmax values from this experiment

> were 0.22 µM and 1.4 pmol/pmol/min,

> respectively.

> Figure 2. Human recombinant aromatase activity as indicated by

fluorimetrically quantified DBF

> dealkylase after treatment with naringenin, chrysin, genistein and dadizein.

Each data point is the

> mean (± SEM) from three separated experiments. Naringenin and chrysin were

effective inhibitors

> of the enzyme with a Ki = 0.3 and 1.0 µM, respectively, while genistein and

daidzein were ineffective

> as shown by a Ki > 50 µM.

>

> 714 K.M. Edmunds et al.

> greater than 99% endometrial stromal cells

> (data not shown).

> Phytoestrogen treatment did not attenuate

> aromatase activity in ESC from women

> with endometriosis (n = 11) at any concentration

> tested (Fig. 3). However, genistein

> (10–9–10–6 M) treatment of ESC from

> women without endometriosis (n = 7) resulted

> in a significant increase in aromatase activity

> (P < 0.05) to approximately 150% above

> the activity observed in untreated ESC from

> the same patient (Fig. 4), whereas daidzein,

> naringenin and chrysin treatment had no

> effect. Furthermore, the genistein induced

> increase in aromatase activity was not attenuated

> by co-treatment with the estrogen

> receptor antagonist ICI 182,780 (P > 0.1,

> Fig. 5).

> 3.3. Immunocytochemistry

> Immunopositive aromatase staining was

> evident as a diffuse brown cytoplasmic precipitate

> (Fig. 6) that was absent in control

> cultures where the primary antibody was

> substituted with non-immune serum. Immunopositive

> staining was focally present in

> some but not all genistein (10–6 M) treated

> ESC from eutopic endometrium of women

> without endometriosis. Moreover, no immunoreactive

> aromatase staining was visible in

> the untreated ESC taken from the same

> patient.

> 4. DISCUSSION

> The objective of the current study was to

> screen dietary phytoestrogens for their ability

> to inhibit human recombinant aromatase

> activity and to determine the effect of dietary

> phytoestrogens on endometrial stromal

> cell aromatase activity in culture. Although

> naringenin and chrysin inhibited aromatase

> in our cell-free assay, they were ineffective

> in endometrial stromal cell cultures from

> Figure 3. Aromatase activity was unchanged in genistein treated endometrial

stromal cell cultures

> from women with endometriosis (n = 11). The control bar represents the

aromatase activity from

> the vehicle treated cells from each of the patients and the data bars

represent the aromatase activity

> of the cells following treatment with genistein represented as percent of

control. The control value

> has arbitrarily been set to 100% and data are presented as the mean ± SEM.

>

> Genistein induced increased aromatase activity 715

> Figure 4. The effects of genistein treatment for 24 h on aromatase activity in

endometrial stromal

> cells obtained from eutopic endometrium of women without endometriosis (n =

7). The control bar

> represents the aromatase activity from the untreated cells from each of the

patients and the data bars

> represent the aromatase activity of the cells following treatment with

genistein represented as percent

> of control. The results are the mean (± SEM) from seven different cultures.

Values with different

> superscripts are significantly (P < 0.05) different.

> Figure 5. The effects of 10–6 M genistein (GEN) alone and in combination with

10–6 M ICI 182 780

> (ICI), and non-selective estrogen receptor antagonist, on aromatase activity

obtained from the eutopic

> endometrium of women without endometriosis (n = 3). The results are the mean

(± SEM) from

> three different cultures. Means identified with a different letter were

significantly different (P =

> 0.008).

>

> 716 K.M. Edmunds et al.

> women with and without endometriosis and

> thus are unlikely to have any potential therapeutic

> benefit in the management of

> endometriosis. In contrast, genistein, the

> dominant isoflavone found in soy-based

> foods, was inactive in the cell-free assay but

> to our surprise increased aromatase activity

> in endometrial stromal cells of women

> without endometriosis. These data suggest

> that the observed effects of genistein are not

> mediated through direct effects of genistein

> on enzyme activity but indirectly via

> enhanced aromatase expression in endometrial

> stromal cells or via intermediates on

> aromatase activity. This point is supported

> by evidence of immunocytochemical staining

> for aromatase in genistein treated but

> not untreated cells. In our study, the concentrations

> of genistein that were used to

> treat the human endometrial stromal cells

> (1 nM to 10 mM) correspond to the serum

> concentrations of both Asian and Caucasian

> women who are consuming soy-based

> foods [27–29] and thus are considered to be

> physiologically relevant. Taken together,

> our results suggest that while phytoestrogens

> may have health benefits such as the

> proposed protection against breast cancer

> development [11, 12], genistein is unlikely

> to have any therapeutic value in the management

> of endometriosis and more importantly

> may increase aromatase activity in

> the endometrium and thus could be an

> important factor in the pathobiology of this

> enigmatic disease.

> In the present study, aromatase was not

> detected by immunohistochemistry in control

> cultures of endometrial stromal cells

> from women without endometriosis. In

> addition, aromatase activity of vehicle

> treated endometrial cells was at background

> levels for the assay and thus supports the

> view that aromatase is either absent or

> inhibited in the endometrium from women

> without endometriosis. Our findings are in

> agreement with prior studies in which aromatase

> cytochrome P450 has been reported

> to be expressed in the endometrium of

> women with endometriosis but is either

> absent [4], or expressed at low levels in the

> endometrium of women without endometriosis

> [30]. Therefore, the patients in the

> current study were grouped into two categories:

> endometriotic and non-endometriotic.

> None of the phytoestrogens tested

> inhibited aromatase activity of the ESC

> from women with endometriosis. However,

> in the current study, genistein-treatment of

> Figure 6. Immunocytochemical staining for aromatase in untreated cells (A) and

cells treated with

> 10–6 M genistein ( reveals positive staining in the treated cells (arrows).

>

> Genistein induced increased aromatase activity 717

> ESC from women without endometriosis

> induced an increase in aromatase activity to

> 150% of the untreated controls similar to

> the findings using adrenocortical carcinoma

> cell lines treated with herbicides [31,

> 32]. Furthermore, our results are harmonious

> with the previous finding that genistein

> (30 µM) increased aromatase activity 3 fold

> in the H295R human adrenocortical carcinoma

> cell line [33]. Hence, genistein treatment-

> induced changes in aromatase activity

> could lead to increased local levels of estrogens

> in the endometrium. However, the

> functional significance of genistein induced

> changes in aromatase activity is unknown.

> A previous study has demonstrated that

> genistein treatment increased cell proliferation

> and was weakly estrogenic in

> endometrial stromal cell and Ishikawa cell

> cultures [34]. However, genistein treatment

> antagonized the effects of estradiol in these

> cultures suggesting that genistein is a competitive

> antagonist of estradiol. Therefore,

> a genistein induced increase in aromatase

> activity and local estrogen production in the

> endometrium could be relevant in hypoestrogenic

> states such as menopause. While

> genistein treatment was without effect on

> the endometrium of macaque monkeys

> with surgically induced menopause [35],

> our proposal is supported by the observation

> that endometrial hyperplasia was significantly

> more prevalent in postmenopausal

> women receiving soy tablets vs. a reference

> group that received a placebo [36]. Moreover,

> a recent study [37] has also shown that

> high dose phytoestrogens can reverse the

> antiestrogenic effects of clomiphene citrate

> on the endometrium. Hence, we propose

> that the effects of soy isoflavones, including

> genistein on the endometrium is complex

> and requires further study.

> The mechanism through which genistein

> treatment increased aromatase activity in

> the endometrium remains unknown. Although

> estradiol has been shown to increase aromatase

> activity in ESC cultures [38], several

> distinct lines of evidence lead us to

> suggest that genistein is not acting through

> an estrogen receptor mediated pathway in

> our cultures to increase aromatase activity.

> Genistein is a preferential estrogen receptor

> (ER)-ß agonist and has been shown to have

> estrogenic actions in a variety of tissues in

> the rat [39, 40]. However, ER-a, not ER-ß

> is the dominant ER sub-type expressed in

> the endometrium [41]. Furthermore, it is

> unlikely that genistein causes stimulation

> of aromatase in the endometrium by acting

> through a functional estrogen receptor pathway

> because we have shown that the stimulation

> of aromatase in ESC by genistein is

> not attenuated by co-treatment of the cells

> with ICI 182,780 which is a non-selective

> estrogen receptor antagonist. We therefore

> propose that it is unlikely that genistein

> stimulates aromatase activity in endometrial

> stromal cell cultures by acting directly

> via the ER. Alternatively, we propose that

> genistein can stimulate aromatase activity

> in the endometrium through inhibition of

> phosphodiesterase activity and result in

> increased levels of cAMP. Support for this

> proposal comes from evidence that genistein

> inhibits cAMP-phosphodiesterase

> activity in a variety of cell types [42–44]. In

> addition, aromatase expression in the

> endometrium is regulated through cAMPinduced

> promoter II [45] and cAMP-treatment

> has previously been shown to result in

> a 26–60 fold increase in endometrial aromatase

> activity [46].

> Soy products are widely believed by the

> public to provide health benefits. The Food

> and Drug Administration has released an

> approval for foods that contain at least

> 6.25 g of soy protein/serving to contain a

> cardiovascular health claim (November 10,

> 1999; No. 279) and this has led to a plethora

> of soy-based and fortified foods as well as

> soy supplements to emerge on to the market

> [47]. Phytoestrogens are efficiently absorbed

> after ingestion and their bioavailability is

> high enough to have biological effects [48].

> Furthermore, contemporary studies reveal

> that non-Asian women are ingesting increasing

> amounts of phytoestrogens in their diet

> as part of a trend towards a healthier lifestyle

> [49, 50]. Despite potential health benefits

> for women of some age groups, we speculate

>

> 718 K.M. Edmunds et al.

> that genistein consumption by women of

> reproductive age may have associated health

> risks. Moreover, epidemiological evidence

> demonstrates that Oriental women have a

> higher incidence of endometriosis than

> Caucasian women suggesting a link between

> endometriosis and dietary phytoestrogens,

> as Asian diets are high in soy isoflavones

> [51, 52]. Hence, consumption of soy products

> by women of reproductive age may not

> be without consequence for endometrial

> aromatase activity and potentially endometriosis.

> In summary, the results of this study

> demonstrate that dietary compounds, such

> as genistein which is present in foods

> including soy milk and tofu that the general

> public views as healthy alternatives to traditional

> foods in the North American diet,

> can increase the local production of estrogen

> in the ESC. Genistein-induced changes

> in endometrial aromatase activity may have

> detrimental effects which could lead to

> increased risk for estrogen-dependent diseases

> which involve the dysregulation of

> aromatase such as endometriosis, adenomyosis

> and uterine leiomyomas.

> ACKNOWLEDGEMENTS

> The authors gratefully acknowledge the technical

> assistance of Louise Beecroft, RN

> and Vasko (Bill) Georgievski.

> This study was supported by funding from the

> Canadian Network of Toxicology Centres

> (WGF) and the Canadian Institutes of Health

> Research (MOP 62681; WGF and ACH).

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> can reverse the antiestrogenic effects

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> on cyclic AMP phosphodiesterase and

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> inhibition of cyclic-AMP phosphodiesterase

> by genistein and tyrphostin 51.

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> tyrosine kinase inhibitor genistein increases

> basal cAMP and potentiates forskolininduced

> cAMP accumulation in A549 human

> airway epithelial cells. Mol Cell Biochem

> 2002, 240: 131–133.

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> ER, Clyne CD. Role of CRE-binding protein

> (CREB) in aromatase expression in breast adipose.

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> E2 stimulates aromatase expression in

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> Endocrinol Metab 1997, 82: 600–606.

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> Nechemias L, Wolfe BE, Brashear WT, Kirschner

> AS, Cassidy A, Heubi JE. Bioavailability

> of pure isoflavones in healthy humans

> and analysis of commercial soy isoflavone

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> LC, Wu AH, Kolonel LN. Association of soy

> and fiber consumption with the risk of

> endometrial cancer. Am J Epidemiol 1997,

> 146: 294–306.

> [50] Guthrie JR, Ball M, Murkies A, Dennerstein

> L. Dietary phytoestrogen intake in mid-life

> Australian-born women: relationship to health

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> [51] Miyazawa K. Incidence of endometriosis

> among Japanese women. Obstet Gynecol

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> and race. Aust N Z J Obstet Gynaecol 1992,

> 32: 164–165.

>

[Guest guest]

INVOLVEMENT AND DYSFUNCTION OF THE

FEMALE REPRODUCTIVE SYSTEM

MYCOSES

Several mycoses can infiltrate the uterus, salpinx, and ovaries

during dissemination45,93,95,174,197-203 (Table 2). Conversely,

isolated genital blastomycosis and histoplasmosis

can be acquired by sexual transmission.199,202 Fungal infections

of female reproductive organs can be asymptomatic

or present as endometritis and/or tubo-ovarian abscess

causing menstrual irregularities, menorrhagia, dysmenorrhea,

anovulation, and/or infertility.197-203

Mycoses can cause dysregulation of the reproductive

endocrine axis via mechanisms other than tissue invasion.

For example, Arora et al204 reported hyperprolactinemiainduced

amenorrhea and galactorrhea in a woman with blastomycosis

involving the pleural cavity. This was attributed

to prolactin production by the chest-wall inflammatory process,

a well-described condition that causes prolactin release.

Furthermore, the Fusarium toxin zearalenone can induce

hyperestrogenism in female mammals but rarely in

humans, leading to decreased LH and progesterone levels,

infertility, vulvovaginitis, and decreased milk production.184

http://www.aspergillus.org.uk/secure/articles/pdfs2/04-08-2008.pdf

I think theres probalby a little more to this than that. or damn, I was exposed

to zea. which I'm really not doughting to much, it may be rare but that doesn't

mean it doesn't happen. something that stands out for me was the plaster in my

victorian home and maybe what was in it,to make it. besides animal hair and

possably straw. plaster can and does absorb water,but it takes a good amount for

it to fall out.

http://en.wikipedia.org/wiki/Phosphodiesterase_inhibitor

I'm seeing a pretty obvious patteren with several things I recently posted.

concerning metabolism disfunction,sensory reception, and the nervous system. and

also while all our organs send and recieve messages from the brain normally,

they still do have their own functions sepertly. and what might be looked at as

playing a big role in sensitivity issues with these organs, rightfully so, there

can still be some differences in whats affecting the organ itself,and may be

dependent on amount of organ damage and what effects come from that. guess what

I'm tring to say is that while we all suffer the same illment, there are

differences based on amount of organ damage to what organs and whats going on in

those organs can affect us differently.

[Guest guest]

and I was tested for these infections,negitive. but I did have some viginal

yeast infections off and on. some pretty bad ones.

Dr. Croft found trichothecenes and smooth muscle growth in my tissues from

hisderectomy.

>

> INVOLVEMENT AND DYSFUNCTION OF THE

> FEMALE REPRODUCTIVE SYSTEM

> MYCOSES

> Several mycoses can infiltrate the uterus, salpinx, and ovaries

> during dissemination45,93,95,174,197-203 (Table 2). Conversely,

> isolated genital blastomycosis and histoplasmosis

> can be acquired by sexual transmission.199,202 Fungal infections

> of female reproductive organs can be asymptomatic

> or present as endometritis and/or tubo-ovarian abscess

> causing menstrual irregularities, menorrhagia, dysmenorrhea,

> anovu

[Guest guest]

letrozole for endometriosis

http://www.endometriosiszone.org/display.asp?page=news_0402_letrozole

AZOLES,ANTIFUNGAL

COMMON ANTIFUNGAL MECHANISM OF ALL AZOLES AGAINST ALL FUNGI, BOTH PLANT AND

HUMAN PATHOGENS

Their mechanism is based on interference with the activity of fungal lanosterol

14á-demethylase, a member of the cytochrome P450 family. Fungal lanosterol

14á-demethylase is responsible for transforming lanosterol to ergosterol, which

is an essential constituent of fungal cytoplasmic membrane. The inhibition of

ergosterol formation would result in fungal cell wall disorganization and,

finally, stop fungal growth. The mode of action of azoles, therefore, is

fungistatic rather than fungicidal.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC90772/

endocrine disruptors- " an exogenous agent that interferes with the

synthesis,secretion,transport,binding,action,or elimination of natural hormones

in the body which are responsible for the maintenance or

homeostasis,reproduction,development and or behavior. " EPA

http://www.programm-mgu.ch/de/home/Forschung/inhouse/Par/01/File/MGU-DUWKolloq-0\

709.pdf

>

> INVOLVEMENT AND DYSFUNCTION OF THE

> FEMALE REPRODUCTIVE SYSTEM

> MYCOSES

> Several mycoses can infiltrate the uterus, salpinx, and ovaries

> during dissemination45,93,95,174,197-203 (Table 2). Conversely,

> isolated genital blastomycosis and histoplasmosis

> can be acquired by sexual transmission.199,202 Fungal infections

> of female reproductive organs can be asymptomatic

> or present as endometritis and/or tubo-ovarian abscess

> causing menstrual irregularities, menorrhagia, dysmenorrhea,

> anovulation, and/or infertility.197-203

> Mycoses can cause dysregulation of the reproductive

> endocrine axis via mechanisms other than tissue invasion.

> For example, Arora et al204 reported hyperprolactinemiainduced

> amenorrhea and galactorrhea in a woman with blastomycosis

> involving the pleural cavity. This was attributed

> to prolactin production by the chest-wall inflammatory process,

> a well-described condition that causes prolactin release.

> Furthermore, the Fusarium toxin zearalenone can induce

> hyperestrogenism in female mammals but rarely in

> humans, leading to decreased LH and progesterone levels,

> infertility, vulvovaginitis, and decreased milk production.184

> http://www.aspergillus.org.uk/secure/articles/pdfs2/04-08-2008.pdf

>

> I think theres probalby a little more to this than that. or damn, I was

exposed to zea. which I'm really not doughting to much, it may be rare but that

doesn't mean it doesn't happen. something that stands out for me was the plaster

in my victorian home and maybe what was in it,to make it. besides animal hair

and possably straw. plaster can and does absorb water,but it takes a good amount

for it to fall out.

>

> http://en.wikipedia.org/wiki/Phosphodiesterase_inhibitor

> I'm seeing a pretty obvious patteren with several things I recently posted.

concerning metabolism disfunction,sensory reception, and the nervous system. and

also while all our organs send and recieve messages from the brain normally,

they still do have their own functions sepertly. and what might be looked at as

playing a big role in sensitivity issues with these organs, rightfully so, there

can still be some differences in whats affecting the organ itself,and may be

dependent on amount of organ damage and what effects come from that. guess what

I'm tring to say is that while we all suffer the same illment, there are

differences based on amount of organ damage to what organs and whats going on in

those organs can affect us differently.

>

[Guest guest]

sorry, wasn't tested for these fungal infections, my head was somewhere else. I

was tested for sexually transmitted diseases. later after exposure at the

second home I was tested and had type 2 candidia al. along with some others.

>

> INVOLVEMENT AND DYSFUNCTION OF THE

> FEMALE REPRODUCTIVE SYSTEM

> MYCOSES

> Several mycoses can infiltrate the uterus, salpinx, and ovaries

> during dissemination45,93,95,174,197-203 (Table 2). Conversely,

> isolated genital blastomycosis and histoplasmosis

> can be acquired by sexual transmission.199,202 Fungal infections

> of female reproductive organs can be asymptomatic

> or present as endometritis and/or tubo-ovarian abscess

> causing menstrual irregularities, menorrhagia, dysmenorrhea,

> anovulation, and/or infertility.197-203

> Mycoses can cause dysregulation of the reproductive

> endocrine axis via mechanisms other than tissue invasion.

> For example, Arora et al204 reported hyperprolactinemiainduced

> amenorrhea and galactorrhea in a woman with blastomycosis

> involving the pleural cavity. This was attributed

> to prolactin production by the chest-wall inflammatory process,

> a well-described condition that causes prolactin release.

> Furthermore, the Fusarium toxin zearalenone can induce

> hyperestrogenism in female mammals but rarely in

> humans, leading to decreased LH and progesterone levels,

> infertility, vulvovaginitis, and decreased milk production.184

> http://www.aspergillus.org.uk/secure/articles/pdfs2/04-08-2008.pdf

>

> I think theres probalby a little more to this than that. or damn, I was

exposed to zea. which I'm really not doughting to much, it may be rare but that

doesn't mean it doesn't happen. something that stands out for me was the plaster

in my victorian home and maybe what was in it,to make it. besides animal hair

and possably straw. plaster can and does absorb water,but it takes a good amount

for it to fall out.

>

> http://en.wikipedia.org/wiki/Phosphodiesterase_inhibitor

> I'm seeing a pretty obvious patteren with several things I recently posted.

concerning metabolism disfunction,sensory reception, and the nervous system. and

also while all our organs send and recieve messages from the brain normally,

they still do have their own functions sepertly. and what might be looked at as

playing a big role in sensitivity issues with these organs, rightfully so, there

can still be some differences in whats affecting the organ itself,and may be

dependent on amount of organ damage and what effects come from that. guess what

I'm tring to say is that while we all suffer the same illment, there are

differences based on amount of organ damage to what organs and whats going on in

those organs can affect us differently.

>

[Guest guest]

http://en.wikipedia.org/wiki/Lanosterol_14_alpha-demethylase

http://en.wikipedia.org/wiki/Cytochrome_P450

> >

> > INVOLVEMENT AND DYSFUNCTION OF THE

> > FEMALE REPRODUCTIVE SYSTEM

> > MYCOSES

> > Several mycoses can infiltrate the uterus, salpinx, and ovaries

> > during dissemination45,93,95,174,197-203 (Table 2). Conversely,

> > isolated genital blastomycosis and histoplasmosis

> > can be acquired by sexual transmission.199,202 Fungal infections

> > of female reproductive organs can be asymptomatic

> > or present as endometritis and/or tubo-ovarian abscess

> > causing menstrual irregularities, menorrhagia, dysmenorrhea,

> > anovulation, and/or infertility.197-203

> > Mycoses can cause dysregulation of the reproductive

> > endocrine axis via mechanisms other than tissue invasion.

> > For example, Arora et al204 reported hyperprolactinemiainduced

> > amenorrhea and galactorrhea in a woman with blastomycosis

> > involving the pleural cavity. This was attributed

> > to prolactin production by the chest-wall inflammatory process,

> > a well-described condition that causes prolactin release.

> > Furthermore, the Fusarium toxin zearalenone can induce

> > hyperestrogenism in female mammals but rarely in

> > humans, leading to decreased LH and progesterone levels,

> > infertility, vulvovaginitis, and decreased milk production.184

> > http://www.aspergillus.org.uk/secure/articles/pdfs2/04-08-2008.pdf

> >

> > I think theres probalby a little more to this than that. or damn, I was

exposed to zea. which I'm really not doughting to much, it may be rare but that

doesn't mean it doesn't happen. something that stands out for me was the plaster

in my victorian home and maybe what was in it,to make it. besides animal hair

and possably straw. plaster can and does absorb water,but it takes a good amount

for it to fall out.

> >

> > http://en.wikipedia.org/wiki/Phosphodiesterase_inhibitor

> > I'm seeing a pretty obvious patteren with several things I recently posted.

concerning metabolism disfunction,sensory reception, and the nervous system. and

also while all our organs send and recieve messages from the brain normally,

they still do have their own functions sepertly. and what might be looked at as

playing a big role in sensitivity issues with these organs, rightfully so, there

can still be some differences in whats affecting the organ itself,and may be

dependent on amount of organ damage and what effects come from that. guess what

I'm tring to say is that while we all suffer the same illment, there are

differences based on amount of organ damage to what organs and whats going on in

those organs can affect us differently.

> >

>