1. 1
Understanding
the
effects
of
steroid
hormone
exposure
on
direct
gene
regulation
T.S.
Wiley1
and
J.T.
Haraldsen2
1
Wiley Compounding Systems, Santa Fe, New Mexico, 87504, USA
2
Department of Physics and Astronomy, James Madison University, Harrisonburg, Virginia, 22802, USA
Steroid
hormones
have
been
widely
overlooked
as
controllers
of
gene
expression.
Through
the
mechanisms
of
gene
expression
(DNA
methylation,
histone
methylation,
and
RNAi),
we
discuss
the
impact
of
normal
reproductive
templates
on
the
pulsatility
and
amplitude
of
potential
gene-­‐regulating
treatment
protocols.
By
examining
the
interactions
of
estradiol
(E2)
and
progesterone
(P4)
in
women,
we
propose
that
changes
in
physiologic
reproductive
hormone
templates
of
exposure
and
timing
can
affect
fertility
and
even
cancer
through
the
silencing
or
amplification
of
gene
products;
such
as
P53
and
Bcl-­‐2
in
women.
We
propose
that
uncontrolled
hormone
levels,
due
to
aging
and/or
the
environment,
may
be
restored
to
a
normal
youthful
template
of
gene
expression
through
the
fluctuating
exogenous
application
of
E2
and
P4
that
mimic
the
normal
hormonal
milieu
of
reproductive
health.
We
hypothesize
that
this
may
lead
to
a
lower
risk
of
the
chronic
illnesses
of
aging
and
a
better
quality
of
life
in
patients
suffering
those
conditions.
Introduction
Steroid hormones are critical to the natural development
of multicellular organisms and complex cellular functions
for all planets, animals, and insects (1-4). Through the use
of receptors and gene products, steroid hormones
constantly control these regulatory systems using iterative
hormone interactions (5,6). Since many diseases can be
traced back to the uncontrolled or silenced gene
expression (7), understanding the mechanisms by which
steroid hormones control or modify gene products is
paramount for the medical community. This is evident in
the well-known use of corticosteroids to reduce
inflammation by silencing inflammatory gene cascades
(8,9).
Since the expression of regulatory genes is critical
for the sustainability of life through the production of
amino acids, enzymes, and proteins (REF), if there is an
error in how and when specific genes are expressed,
steroid hormones can either produce or reduce the levels
of functionality down stream of all gene products
throughout the body (7,10). This leads to body-wide
disarray in all the cells’ ability to regulate all gene
expression, which has been observed in cancer patients as
well as other instances of chronic illnesses in aging (11),
such as heart disease, diabetes, and neuro-degenerative
states (12-16).
Although gene expression is controlled through
many different mechanisms in the body, the three most
prominent and recognized mechanisms today are through
DNA (deoxyribonucleic acid) methylation (17), histone
methylation and acetylation (18), and RNA (ribonucleic
acid) interference (RNAi) (19). These mechanisms are
critical for maintenance of gene function during the
replication and transcription processes (17-19), where they
control gene expression at different levels:
• DNA methylation controls genes through a direct
tagging of specific cytosine bases on the DNA
strand with a methyl group (CH3) (17).
• Histone methylation and acetylation through
chromatin action allows histones to reduce or
increase gene expression by literally stopping the
transcription process. This acts as a locking
mechanism on the DNA strand (18).
• RNAi is a completely different mechanism that
involves the literal cutting and splicing of
individual small pocket DNA strands (sDNA) to
turn genes on and off or insert new material (19).
While these mechanisms differ in their methods, we show
that all three of these mechanisms have a striking
commonality; they are controlled primarily through
interactions with steroid hormones.

2. 2
Since steroid hormones (i.e. estradiol (E2),
progesterone (P4), testosterone (T), cortisol (C)) control
many gene products (20-23), it is important to examine
how the loss or disarray of this hormone product can be
manifested as illness. By understanding the potentials of
gene regulation within a cell, we can discuss the general
mechanisms by which changing steroid hormone levels
may have a distinct effect on the expression and
repression of genes, which can in turn lead to an increased
risk of the diseases of aging.
In this paper, we examine the connection of
hormone production and deterioration, from aging and/or
environmental changes, to the gene regulation through the
various methods (cited above) of gene silencing and
amplification. Since steroid hormones control the cellular
regulation of proliferation and apoptosis, it is apparent that
the disruption of these hormones can systematically lead
to diseases like diabetes, Alzheimer’s, and cancer through
an increase or decrease via the methylation of DNA,
uncontrolled histone modifications, and/or effects on the
transcription of RNAi. Through an examination of specific
hormonal processes, we elucidate the effects of aging and
environmental cues on the production E2 and P4 that may
ultimately disrupt the regulation of actual gene products.
Through a detailed examination of the known literature in
the field, we hypothesize that the pulsatility (exposure
frequency) and amplitude (varying amounts) of steroid
hormones will affect the gene expression via the three
known mechanisms of DNA methylation, histone
methylation and acetylation, and RNA interference.
In this paper, it is our intent to show how steroid
hormones may be used to control these mechanisms of
regulation through changes in exogenous dosing and
timing of administration. Below, we provide a description
of the steroid hormones, an overview of the gene
expression mechanisms, and a discussion of the
implications of pulsatility and amplitude in younger
female reproductive templates, which varies significantly
from the current standard of care replacement therapy,
static non-varying dosing of synthetic hormone-like
pharmaceuticals.
Background
The expression or repression of genes may happen in a
number of ways. Currently, the known mechanisms for
gene regulation being studied are via DNA methylation,
Histone methylation and acetylation, and RNAi splicing
(17-19). As mentioned above, these mechanisms provide
specific actions on the genes to regulate gene products by
altering the DNA transcription processes.
It has been long ignored that steroid hormones
production is the response to the environment that has a
direct affect on all three mechanisms. This makes steroid
hormones likely candidates for study in genomics and
epigenetics.
Steroid Hormones
Steroid hormones are involved in all genetic, cellular, and
bodily processes (2-4). They are growth and reproduction
catalysts in bone, brain, heart, and reproductive organs
(24). The production of estrogen in the body of a young
female is critical for the regulation of functional gene
control (25). Since estrogen, as a steroid hormone, affects
gene expression (26), it is important to understand the
Figure
1
-­‐
(a)
The
normal
estradiol
and
progesterone
cycles
throughout
a
women’s
menstrual
cycle
(30).
The
day
12
peak
in
estradiol
correlates
to
increase
in
Bcl-­‐2
and
a
decrease
P53
gene
products,
while
the
day
21
peak
in
progesterone
correlates
to
a
decrease
in
Bcl-­‐2
and
an
increase
P53.
These
signal
the
proliferation
and
apoptosis
of
cells
throughout
the
cycle.
(b)
The
E2
and
P4
dosing
template
of
the
Wiley
Protocol
proposed
to
mimic
the
normal
cycle
using
bio-­‐identical
hormone
replacement
therapy
through
transdermal
application.

3. 3
possible methods by which steroid hormones act on these
mechanisms.
Steroids travel through the blood using lipid
carriers. This is critical for protein production within the
cell that will, in turn, create hormone receptors for various
other steroids (27). Once in a membrane receptor, the
steroid will detach from the carrier and translocate through
the cell membrane, where it can either directly access the
nuclear promoter regions or bind to a non-nuclear
receptor. Once the hormone-receptor complex has
phosphorylated down to the nucleus (28), it will bind to
those appropriate specific hormone response elements and
promoter regions of the DNA strand. The interaction
between the steroid hormone-receptor complex and
binding sites causes the DNA signals to for messenger
RNA (mRNA) transcription, which results in protein
synthesis outside the nucleus, often other steroid and/or
hormone receptors (29). All cellular and bodily functions
are affected by the production of these proteins, which
means that the conscious well being of a host is highly
dictated by the amplitude and frequency of steroid
hormones.
The body is accustomed to a regular schedule for
steroid hormone exposure (26). This is clearly evident in
the estrogen and progesterone cycles of females across all
species. The female menstrual cycle is a complex
interaction of estrogen and progesterone through
fluctuating repetitive patterns over 28 days (shown in Fig.
1(a)) (30,31). As a woman ages and her egg base declines,
the normal patterns are disrupted and eroded leading to an
erratic peri-menopausal state preceding menopause. In this
state, literature verifies there are significant increases a
woman’s risk of heart disease, diabetes, reproductive
cancers, and osteoporosis (26).
It is the ripening of eggs that creates the standard
estradiol cycle in the reproductive template, which
produces a large peak of estrogen at approximately the
12th
days of a woman’s cycle (31,32). This surge of
estrogen provokes a progesterone receptor from the
nucleus of the cell as well as spikes the luteinizing
hormone in the brain to instruct the ovary’s release of the
egg. It is the ensuing degradation of the ruptured corpus
luteum that begins the progesterone cycle (P4) at this time.
P4 peaks on day 21 of this menstrual cycle. Should
fertilization occur between days 19-22, the women will
continue to experience even higher levels of progesterone
in the now established pregnancy. However, if fertilization
does not occur, apoptosis begins and the hormonal cues
instruct the shedding of the uterine lining. This action of
P4 blocks E2 receptors to assure repopulation on day 29/1
of the new cycle to restart the template.
To understand how the time and amount of
hormonal exposure affects the overall expression of genes,
we move now to discuss the cited mechanisms that control
gene expression and how steroid hormones, especially
estradiol, ties into these mechanisms.
DNA Methylation
The methylation of DNA is a process in which a methyl
group (CH3) bonds to the 5 position of cytosine (C) only
(33). This process is critical for the development of
organisms due to its control over cellular differentiation,
and its ability to alter gene expression over evolution.
The methylation occurs at cytosine sites that are
connected to guanine (G) sites through the phosphate
backbone, denoted as CpG (34), in the linear sequence of
DNA bases. This is different than the hydrogen bonding
that occurs between DNA base pairs (C-G and adenine
(A) –thymine (T)). Methylation of non-CpG bases can
occur, but has only been found, interestingly, in stem
cells. The process of DNA methyltransferases (DNMT1
and DNMT3) transferring methyl groups to DNA using S-
adenosyl methionine as a donor (illustrated in Figure 2),
which is mitigated through the interactions of steroid
hormones (17).
The percentage of methylated DNA in the body is
known to vary more in the first stages of life, but seems to
= CpG
= CH3
Promoter Gene Anti-Oncogene
NormalCancerous
= S-adenosyl methionine
Figure
2
–
General
DNA
Methylation
mechanism.
The
process
of
DNA
methyltransferases
(DNMT1
and
DNMT3)
transferring
methyl
groups
to
DNA
using
S-­‐adenosyl
methionine
as
a
donor,
which
is
mitigated
through
the
interactions
of
steroid
hormones.

4. 4
stabilizes quickly (34). Once stabilized, enzymes move
along DNA and maintain the level of methylated cytosine
sites throughout reproductive life. This is evident through
epigenetic silencing and amplification of various genes
(35,36).
Over the past decade, it has been discovered that
hypermethylation (increased methylation) or
hypomethylation (decreased methylation) can occur,
specifically in the later stages of life when hormone
production and interactions decline in both men
(testosterone) and women (estrogen). Hypomethylation
has been linked to the over-expression of oncogenes in
cancer cells. However, in the literature, the exact
controlling mechanism still remains unclear.
For example, in a 2001 paper by Hartsough et al
(37), a clear link between DNA methylation and the
proliferation of cancer cells was discovered. This is an
indication of the distinct importance of the need to
understanding these mechanisms. For breast cancer
patients, the loss of steroid hormones is critical in the
silencing of crucial gene products (38,39). The fact that
the use of synthetic hormones does not alleviate this
silencing factor is a note of great importance (40,41). The
question must be asked, do synthetic hormones affect
these mechanisms of regulation in any normal way?
Histones
Histones are proteins that affect the compaction and
contraction of DNA using tightly wound bundles called
nucleosomes. Histones, through chromatin, control the
winding and unwinding of DNA for transcription
controlling the expression of the gene (18). When DNA
needs to be transcribed, the histones unravel, allowing the
DNA transcription process to occur. In this way, histones
play a critical role in gene regulation and expression.
Steroid hormones can control gene activation
through methylation of the histone (similar to DNA
methylation) or through acetylation of the histone in
which an acetyl group (CH3O) is attached (illustrated in
Figure 3). Therefore, the way steroid hormones control
histone action is through methyl and acetyl group (18).
The methylation and acetylation processes can stop the
histone’s ability to unwind the DNA for transcription,
which in turn stops gene expression. This steroid action
effectively silences the histone itself and locks the DNA
from transcription.
When DNA wraps around a histone, that section
of DNA becomes inaccessible and the gene is no longer
active. Often, histone regulation is controlled through
interactions with peptides hormones as well. However,
since histone methylation is dependent on interactions
with methyltransferases (similar to DNA methylation),
steroid hormone interactions are critical (42).
RNA Interference (RNAi)
RNA interference (RNAi) is the inactivation of genes
during the transcription process (19,43,44). This occurs
when microRNA (miRNA) and small interfering RNA
(siRNA) bind to sections of messenger RNA (mRNA) and
stops the production of the gene products, proteins and
enzymes. The siRNA, which is can be as small as 22
nucleotides, are cut from longer double stranded RNA
(dsRNA) by endoribonuclease enzymes called Dicers.
RNA silencing is a critical process in eukaryotic
cells (plants, animals, and fungi)(45,46), because it can
easily stop or enhance the production of vital gene
products for functional or even evolutionary purposes. The
silencing of gene products through RNAi is critical for an
organism’s protection against viral infections or even
cancer. The under or over expression of various gene
products creates an instability in the cell. For more
information on RNAi mechanisms, please refer to
reference 47.
RNAi while capable of silencing and voicing
genes is also (48,49), interestingly, instrumental in
creating steroid production as well as, which, in turn, these
Figure
3
–
Histones
methylation
and
the
activation
of
DNA.
Histones
methylate
through
histone
methyltransferases
using
the
S-­‐adenosyl
methionine
as
a
donor,
which
places
methyl
groups
on
the
H3
and
H4
histones.
Acetylation
typically
uses
an
interaction
with
acetyl-­‐coenzyme
A
(18).
= CH3
= CH3O
Methylated)
Histone)
Acetylated)
Histone)
= S-adenosyl methionine
= Acetyl-Coenzyme A
Ac1ve)DNA)
Inac1ve)DNA)

5. 5
steroids may methylate by histones and regulate gene
protein production in endless cascades.
Hypothesis
It is our hypothesis that gene expression is controlled, not
just by the shear presence of steroid hormones in the
blood, but by the frequency and amplitude of hormone
production within the body. Steroid hormones are known
to control the cell’s regulatory systems through
modification of gene expression. It is also well known that
all steroid hormones follow consistent rhythmic patterns
of production driven by environmental cues. When these
templates are disrupted by aging, pharmaceutical, or
environmental interference, they may shutdown or reverse
gene expression. We further hypothesize that this may
lead to the diseases of cancer, heart disease, diabetes, or
osteoporosis in aging. Below, we elucidate by example
some of these effects of deregulated hormones,
specifically in women (estradiol and progesterone). We
also propose a basic genomic experiment that will confirm
how the pulsatility and amplitude of hormone dosing can
affect the activation of specific regulatory gene products
such as BCL-2 and P53.
It is our belief that the deviation from the normal
rhythmic template of exposure to steroid hormones in men
and women can cause a regulatory chaos on the cellular
level via the 3 mechanisms outlined above, which
manifests itself in many ways including breast cancer and
other peri-menopausal symptoms. Because of the nature of
steroid hormones, static dosing of synthetic hormones-like
drugs (the standard of care in Hormone Replacement
Therapy) does not take into account natural rhythmic
production, which provide cells with the proper signaling
to produce gene products necessary for homeostasis.
Discussion: Genomic consequences of
eroded estrogen cycles
Since steroid hormones are critical to gene regulation, we
focused on just estrogen as an example of the major
consequences of eroded hormone cycles. The menstrual
cycle in women produces greatly varying levels
(amplitudes) of hormones (specifically estrogen and
progesterone) in a pulsating fashion over 28 days. These
hormones affect gene products through methylation of
DNA or histones or through gene silencing via RNAi
(Figures 2 and 3). Since the onset of peri-menopause
signals wild fluctuations in estrogen production, it is
reasonable to assume that the ensuing control of gene
expression also deeply affected adversely (shown in Fig.
4).
The normal cycles of estrogen and progesterone
manifest predictable regulatory gene expression through
the repression or expression of genes like P53 and Bcl-2
and their gene products, which are timed throughout the
cycle as well as initiating cascades of other functional
gene products. As shown in figure 1(a), the normal cycle
consists of an estrogen peak on day 12. This produces an
increase in Bcl-2 (!) until day 12 and a decrease in P53
("). On day 21, the progesterone peaks and commands
the cells to begin apoptosis and signals a decrease in Bcl-2
(") by blocking E2 and an increase in P53 (!). This
allows for regulation and control of the increase and
decrease of cells all over the body.
The hormone cycle signals cells to proliferate and
when apoptosis should occur for normal functioning. As a
woman enters peri-menopause, these signals can be
interrupted due to the body’s inability to produce normal
estrogen and progesterone peaks. Therefore, the body’s
clinical response is one of chaos, which produces hot
flashes, mood swings, irritability, and loss of libido.
However, physically the signals regulating proliferation
and apoptosis have also deteriorated, which can lead to
uncontrolled cell growth known as cancer or osteoporosis.
Studies show that static replacement of hormones
is unsuccessful in the long term in helping women with
symptoms as well any amelioration of the disease process
in aging. These studies have also shown that static dosing
can accurately increase cardiac risk. Therefore, we
conjecture that by following the body’s normal and
Childhood
Puberty
Peri-
menopause
Menopause
Reproductive Years
Estrogen Levels
Relative Cancer Rate
0 60504012 13 Age (years)
Figure
4
–
An
illustration
of
the
estrogen
in
urine
and
relative
cancer
rate
as
a
function
of
age
[52].
As
age
increases
and
a
woman’s
estrogen
level
begins
to
decrease,
her
relative
cancer
risk
begins
to
increase
as
gene
expression
becomes
irregular.

6. 6
evolved rhythmic production template with bio-mimetic,
bio-identical therapy, the body may regain the normal
expression of gene products that may defend against cell
abnormalities.
We refer to the only bio-mimetic, bio-identical
Hormone Replacement Therapy available (The Wiley
Protocol®
) (50), because it is such a regimen that takes
these normal templates of reproductive fitness into
account. The reestablishment of this well-established
template of hormone production should control gene
expression as well. Shown in figure 1(b), the Wiley
Protocol®
provides women with transdermal hormone
therapy that consists of estrogen and progesterone
provided topically. Recent unpublished studies of this
regimen have demonstrated that women have an increase
in sleep, energy, bone growth, libido, and overall quality
of life with no increase in cancer incidence (51). Further
clinical investigation is warranted.
Proposed Clinical Experiment
To examine how hormone regulation will control gene
expression, we intend to examination how of the
activation of gene products for various groups of women
(young, peri-menopausal, post-menopausal) on different
degrees of hormone replacement therapies (static, bio-
mimetic, and none).
The examination of various groups of women
divided into youth, post-menopausal, and Wiley
Protocol® will be subject to blood spot gene identification
on days 12 and 21 of the normal cycle for young and
Wiley Protocol® women. Post-menopausal women will
follow chosen cycle days 12 and 21, respectively.
Using the blood spot gene identification, we can
examine the expression of P53 and BCL-2 for comparison
to the expression in normally reproductive subjects. In
particular, we expect that one should specifically look at
the expression and repression of P53 and BCL-2.
Conclusion
Overall, we propose a hypothesis that steroid hormones
affect gene expression through changes in both amplitude
and frequency of dosing or exposure. A key example of
this would be through the changing of hormones in
women through menopause. Advances in hormone
replacement therapy provide a wide range of testable
parameters for this claim. We suggest the investigation of
the expression of P53 and Bcl-2 in both peri- and post-
menopausal women.

7. 7
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