Identifying How Environmental Chemicals Affect LH Secretion: A Systems Biology Approach

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Identifying How Environmental Chemicals Affect LH
Secretion A Systems Biology Approach Jerome M.
Goldman, Susan C. Laws, Tammy E. Stoker, Michael
G. Narotsky and Ralph L. Cooper U.S.
Environmental Protection Agency, Office of
Research and Development, National Health
Environmental Effects Research Laboratory Endocrin
ology Branch, Reproductive Toxicology Division,
Research Triangle Park, NC
Approaches / Methods Employed
Is there a direct effect of the compound on the
pituitary?
A variety of toxicants have been found to
suppress the surge via an impact on one or more
components of the hypothalamic-pituitary-ovarian
axis. Identification of these targets sites,
using both in vivo and in vitro approaches, has
been the focus of efforts in this ongoing
research project.
Results/Conclusions
If cyclicity is affected, does compound block the
LH surge?
Science Issue
A growing number of environmental toxicants
having an adverse effect upon reproductive
function in the mammalian female have been found
to suppress the LH surge (and ovulation) through
an impact on one or more components of the
hypothalamic-pituitary-ovarian axis. A variety
of approaches have been employed to understand
the site and mechanisms of insult to this axis.
The information will contribute in a meaningful
way to the modeling of such effects and provide a
sound basis for reproductive risk assessment.
This project is designed to provide methods and
models to determine the neuronal/molecular
targets that can be used for risk assessment of
environmental toxicants that alter the
hypothalamic/pituitary control of gonadal
function, a coordinated physiological system that
regulates reproductive processes across all
mammalian species.

• Toxicant-induced Alterations in the LH Surge
  Assessment of Multiple Target Sites within the
  Hypothalamic-Pituitary-Ovarian Axis.
• Summary of steps in the decision tree
• Are there alterations by individual toxicants
  in estrous cyclicity? Assessment by vaginal
  lavage, ovarian histology.
• If cyclicity is altered, what are the effects
  on the LH surge? Intact or ovariectomized/steroid-
  primed females.
• Is the brain a target site for a toxicant
  suppression of the surge?
• Is there an impact on GnRH secretion? (meausre
  GnRH, GnRH pulses)
• Are hypothalamic neurotransmitters
  affected? Inclusion of work with cell lines
  (adrenal pheochromocytoma PC12 for
  neurotransmitters, GT1-7 neuronal cells for GnRH
  release) as appropriate.
• Is there a direct toxicant effect on pituitary
  hormonal secretion? Use of pituitary perifusion.
• If the target site doesnt appear to be the
  brain, are there effects on steroid hormone
  synthesis and subsequently feedback to the brain
  and pituitary?
• Evaluate steroidogenesis in follicles or ovarian
  micro-injections.
• Toxicant-altered segments of the regulatory
  cascade as homologous with pre-surge events in
  the human. How may any such findings contribute
  to the process of risk assessment?

Research Goals
From Cooper et al., 2000
Atrazine (0 100 mM) added to rat pituitary
tissue fragments in vitro l Failed to affect
the stimulation of LH by gonadotropin releasing
hormone (GnRH). l Indicated that previously
observed suppressive effects of atrazine on the
LH surge were not attributable to a direct action
at the level of the pituitary, but were likely
due to an impact on the hypothalamic mechanisms
responsible for stimulating the surge.
Cooper et al., 2000
The overall research goal is an attempt to
understand the network of physiological processes
whose alterations underlie the impact of
toxicants on reproductive function. Environmental
toxicants are investigated for their ability to
suppression the preovulatory surge of luteinizing
hormone that stimulate the final stages of
oocytic and follicular maturation. An emerging
focus is on the brain as a target site for
reproductive toxicants. If subsequent research
demonstrates that toxicants do indeed target the
hypothalamic regulation of the LH surge, animals
will be assessed at different times over the
morning and early afternoon of vaginal proestrus
to assess the impact of toxicant exposure on the
temporal sequence of changes that leads to a
triggering of the surge. This approach is
designed to identify more clearly the specific
events targeted by individual toxicants that
underlie a suppression of the functional
preovulatory rise in LH.
Impact and Outcomes
What has become quite obvious in assessing the
effect of toxicants on reproductive function is a
need to consider the impact of toxicants within a
physiological system comprised of the
hypothalamus, pituitary and gonads, a system that
operates in a coordinate, regulatory manner to
insure reproductive success. Any of these
components targeted by a toxicant can have an
adverse impact on the LH surge, an event that
serves as a functional endocrine trigger for the
process of ovulation. In addition, the effects
of such endocrine dysregulation may extend beyond
reproductive activity and include tumorigenic
growth and even potential effects on brain
impairments such as Alzheimers disease (Webber
et al., 2004).
Effects of 2 daily oral doses of the
dithiocarbamate pesticide Ferbam on the LH surge
in ovariectomized / estradiol implanted rats. l
Suppression in the surge peak and the area under
the curve for samplings at 1400, 1600, 1800 and
2000h.
Could a blockade of the LH surge be due to
suppression in steroid feedback to brain /or
pituitary?
Is thebrain a target site? If so, is there an
effect on the pulsatile release of LH?
From Goldman et al., 1997

• Unilateral micro-injections of the
  dithiocarbamate pesticide, sodium
  dimethyldithiocarbamate (DMDC, 0.3 mM/ovary)
  under the ovarian bursa (intrabursal, IB)
  surrounding the ovary.
• l Blocked ovulation in the injected ovary (I),
  but not in the non-injected ovary when given at 2
  different times on the day of proestrus. l
  Blocked ovulation from both ovaries when given at
  1900h on diestrus II. l Ovulation on the
  non-injected side was reinstated when
  subcutaneous injections of hCG (1900 h, diestrus
  II) were given, indicating that the blockade in
  ovulation on this side was due to a suppression
  in the LH surge via an effect on ovarian feedback
  to hypothalamus /or pituitary. The ovulatory
  block on the injected side was due to direct
  ovarian effects.

Future Directions
Stoker et al., 2004
One of the more poorly explored areas in
toxicology is an assessment of the brain as a
target site for reproductive toxicants. As a
preponderance of the pre-surge hypothalamic
mechanisms are homologous between the rodent and
human, an identification of the impact of a
toxicant on the cascade of these changes will
provide critical data needed to address issues in
risk assessment. Future research efforts will
serve to characterize how these events are
affected by various reproductive toxicants found
to adversely alter central nervous system
function.
Effects of molinate on the pulsatile secretion of
LH in 3 ovariectomized rats. l Attenuation of the
pulses indicates an impact on the brain
mechanisms regulating the secretion of
gonadotropin-releasing hormone.
Will Administration of a compound alter estrous
cyclicity?
Is an attenuation in GnRH pulsatility
attributable to an effect on the synthesis of
hypothalamic catecholamines?
Future Directions
If the brain is identified as a target site, the
specific events targeted by a toxicant will be
investigated by determining effects on the
temporal sequence of hypothalamic and serum
endocrine changes preceding the generation of the
LH surge.

• The hypothalamic-pituitary-gonadal axis
  constitutes a biological system that regulates
  mammalian reproductive function and impacts a
  widely diverse number of other functional
  processes.
• l In the female, the midcycle surges of the
  gonadotropins, follicle-stimulating hormone (FSH)
  and luteinizing hormone (LH) stimulate the
  developmental recruitment of a new crop of
  follicles and trigger the final stages of oocyte
  and follicular maturation prior to ovulation.
  Both are key to successful reproductive activity.

References
Balchak, SK, Hedge, JM, Murr, AS, Mole, ML,
Goldman, JM. Reproductive Toxicology 14533,
2000. Cooper, RL, Stoker, TE, Tyrey, L, Goldman,
JM, McElroy, WK. Toxicol. Sci. 53297,
2000. Das, PC, McElroy, WK, Cooper, RL. Life
Sci. 733123, 2003. Goldman, JM, Stoker, TE,
Cooper, RL, McElroy, WK, Hein, JF. Neurotoxicol.
Teratol. 16257, 1994. Goldman, JM, Parrish, MB,
Cooper, RL, McElroy, WK. Reprod. Toxicol. 11
185, 1997. Stoker, TE, Perreault, SD, Bremser,
K, Marshall, RS, Murr, A, Cooper, RL. Toxicol.
Sci. (in press). Webber, KM, Bowen, R,
Casadesus, G, Perry, G, Atwood, CS, Smith, MA.
Acta Neurobiol. Exp. 64113, 2004.
Dose-related perturbation of rat estrous
cyclicity by the drinking water disinfection
by-product dibromoacetic acid in response to 2
week gavage exposures. 1/8 height columns-
diestrus, ½ height columns- proestrus, full
height columns- estrus.
From Goldman et al., 1994
From Das et al., 2003
The fungicide metam sodium (MS) was found to
block the LH surge in ovariectomized/ estradiol
(E2)-primed rats via a likely suppression in
dopamine-b-hydroxylase, thereby decreasing
hypothalamic concentrations of the product
neurotransmitter norepinephrine and elevating its
dopamine precursor.
The herbicides atrazine and simazine, previously
shown to block the LH surge, were found to
suppress catecholamine synthesis and
dopamine-b-hydroxylase in PC12 cells over the
course of 24 hours.
Animals are killed at intervals over the day of
proestrus to assess effects of toxicant exposure
on progression of hypothalamic events preceding
the LH surge.
Similarity of the midcycle surge of LH in women
and female rats relative to ovulation. A
suppression in the surge will block the ovarian
events stimulated by this sharp rise in LH and
block ovulation.
From Balchak et al., 2004