The Toxicologist - Society of Toxicology

Results: MC was variable within and between females. Mean and median cycle
lengths of all individual cycles was 30.8 and 30 days (range 17 to 63 days), and approximately
94% of the MC were between 22 to 38 days. Approximately 90% of
SC was within ±20% of the BC. The peak E2 and P4 concentrations were observed
on menstrual days (MD) 13 and 23, respectively, in normal MC. These peaks delayed
in prolonged MC (MD34 and MD59, respectively). Interval between E2 and
P4 peaks, and interval between E2 peak and the next menses were extended in prolonged
cycles while change in interval between P4 peak and next menses was minimal.
Conclusion: Normal range of menstrual cycle in cynomolgus monkey was
from 22 to 38 days. Over time, the normal menstrual cycle can fluctuate up to 20%
from the baseline cycle in individual animals. In prolonged cycles, occurrences of
peak E2 and P4 concentration also delayed but the interval between peak P4 and
onset of the next menses was not affected.
1062 ESTROGENIC IMPACTS OF A STANDARD RODENT
CHOW DIET ON RAT REPRODUCTION AND
DEVELOPMENT.
H. Aoyama 1 , K. L. Takahashi 1 , N. Endo 1 , A. Sato 1 , M. Araki 1 , M. Harigae 1 ,
M. Mitsutani 1 , C. Urakawa 1 , T. Nakamura 1 , N. Sakai 1 , K. Sakasai 1 , S.
Teramoto 1 , F. Horio 2 , A. Murai 2 , M. Kobayashi 2 and H. Hojo 1 . 1 Toxicology
Division, Institute of Environmental Toxicology, Joso, Japan and 2 Graduate School of
Bioagricultural Sciences, Nagoya University, Nagoya, Japan. Sponsor: M. Kuwagata.
Standard rodent chow diets usually contain a considerable amount of phytoestrogens
and are suspected of having unexpected impacts on reproduction and development
of laboratory rats and mice. The present study aimed at examining possible
estrogenic properties of standard chow diets on reproductive and developmental
phenotypes of intact experimental animals, in which inbred Wistar Kyoto
(WKY/NCrlCrlj) rats, 24/sex/group, were fed a purified diet (AIN-93G) containing
no detectable levels of phytoestrogens or a phytoestrogen-rich standard rodent
chow diet (MF) for two successive generations. Parental rats exhibited no significant
differences between the two groups in such parameters as mating, fertility and
delivery indices. However, the numbers of implants and/or newborns per females,
as well as the weights of seminal vesicles and prostates of males in the MF group
were significantly lower than those in the AIN-93G group in both generations.
Significant differences were also found in developmental parameters of F1 and/or
F2 offspring between the two groups: female weanlings in the MF group had significantly
heavier uterus than those in the AIN-93G group, vaginal opening of females
was slightly accelerated in the MF group, and preputial separation of males
was clearly delayed in the MF group. Most alterations in the MF group were similar
to those observed in the animals treated with estrogenic compounds such as 17β
estradiol, ethinyl estradiol and methoxychor. As the total concentration of phytoestrogens
was more than 400 ppm in the MF diet while less than the detectable
limit in the AIN-93G diet in the present study, the differences between the two
groups are suggested to be due, at least in part, to estrogenic effects of phytoestrogens
in the standard rodent chow diet.
Supported by Grant #0901 from the Food Safety Commission of Japan.
1063 EFFECTS OF NEONATAL BISPHENOL-A EXPOSURE
ON PERIPUBERTAL DEVELOPMENT OF FEMALE RAT
HYPOTHALAMUS.
S. M. Losa, K. L. Todd, K. McCaffrey, M. Radford and H. B. Patisaul. Biology,
North Carolina State University, Raleigh, NC.
We and others have previously found that Bisphenol A (BPA) exposure during the
neonatal period, a critical window of sexually dimorphic hypothalamic differentiation
in the rat, can alter the timing of female pubertal onset, induce irregular or premature
cessation of the estrous cycle, and ovarian malformations in adulthood. It is
now well recognized that hypothalamic neurons which produce the kisspeptin family
of peptide hormones (KISS neurons) are critical for initiating puberty and maintaining
estrous cyclicity. Here we explored the hypothesis that neonatal BPA exposure
disrupts the sex specific density of KISS neuronal fibers in the antereoventral
periventricular nucleus (AVPV) and arcuate nucleus (ARC), and the ontogeny of
KISS inputs on gonadotropin releasing hormone (GnRH) neurons. To test this we
used a transgenic strain of Wistar rats where their GnRH neurons produce green
fluorescent protein (GFP). Pups were exposed via subcutaneous injection to vehicle,
17β estradiol (E2), 50μg/kg BPA (the current reference dose) or 50mg/kg BPA
(the current LOAEL) daily from the day of birth (post natal day (PND) 0) through
PND 3 and then cohorts were sacrificed on PNDs 17, 21, 24, 28, and 33 (5-8 animals
per age per exposure; males collected on PNDs 21 and 33) via pericardial perfusion.
Vaginal opening, body weight, serum luteinizing hormone levels and ovarian
histology were also assessed in the females. KISS fiber density and contacts on
GnRH neurons were quantified using immunohistochemistry and confocal microscopy.
Vaginal opening was slightly advanced by E2 and the low dose of BPA. By
PND 33, E2 and 50mg/kg BPA exposed females had AVPV KISS fiber density
more typical of males and decreased numbers of KISS contacts on GnRH neurons.
E2 additionally resulted in decreased ARC KISS fiber density at all time points.
These findings suggest that neonatal BPA exposure can defeminize the female hypothalamus
in the peripubertal window.
1064 IMPACT OF NEONATAL BPA EXPOSURE ON SEXUALLY
DIMORPHIC GENE EXPRESSION IN THE POSTNATAL
RAT HYPOTHALAMUS.
J. Cao, J. A. Mickens, K. McCaffrey, K. L. Todd and H. Patisaul. Biology, North
Carolina State University, Raleigh, NC. Sponsor: G. LeBlanc.
In rats, the first few days of neonatal life are a critical time of sex specific hypothalamic
differentiation. It has been hypothesized that exposure to Bisphenol A (BPA)
during critical windows of development, interferes with sexual differentiation of the
rodent hypothalamus. To test this, we first mapped the temporal and sexually dimorphic
neonatal expression profiles of the two major estrogen receptor isoforms
(ERα and ERβ) in the anterioventral periventricular nucleus (AVPV), medial preoptic
area (MPOA), ventromedial nucleus (VMN) and arcuate nucleus (ARC),
using in situ hybridization. ERα expression was generally higher in females (Long
Evans rat) through postnatal day (PND) 19 except in the ARC. AVPV ERβ expression
was higher in males at birth, then declined and was ultimately higher in females
on PND 19. VMN ERβ levels were higher in females until PND 19. Thus,
we determined that hypothalamic sex differences can be pronounced at birth but
transient, and region specific. We next tested the hypothesis that exposure to BPA
during this period would alter this sexually dimorphic gene expression profile. Long
Evans neonates were exposed to vehicle, 10 μg estradiol benzoate (EB), 50 mg/kg
BPA or 50 μg/kg BPA by subcutaneous injection on the first three days of life
(PNDs 0, 1 and 2). Pups were sacrificed and the brains collected on PNDs 4 and 10
(6-8 per age per sex per exposure group). EB exposure significantly decreased ERα
expression levels in the female AVPV and the MPOA of both sexes. In contrast,
BPA exposure produced the opposite effect in the AVPV and enhanced ERα expression
in both sexes, augmenting the sex difference on PND 4. No effect of BPA
was observed on MPOA ERα expression in either sex. Our results indicate that
BPA may interfere with sexually dimorphic ER expression during the neonatal critical
period. Moreover, the opposite effect of EB and BPA on ERα expression indicates
that BPA may disrupt neuroendocrine differentiation by a mechanism other
than simply binding to ERs and acting as an estrogen mimic.
1065 BENZO(A)PYRENE-INDUCED OVARIAN
DYSFUNCTION RESULTS FROM SUPPRESSED E 2
SYNTHESIS.
A. E. Archibong 1 , A. Ramesh 2 , M. S. Niaz 2 and D. J. Alcendor 3 . 1 Physiology,
Meharry Medical College, Nashville, TN, 2 Biochemistry & Cancer Biology, Meharry
Medical College, Nashville, TN and 3 Microbiology and Center for Health Disparities
Research in HIV, Meharry Medical College, Nashville, TN.
Epidemiological data suggest that smokers have delayed conception and premature
ovarian failure leading to early menopause. The toxic effects of cigarette smoke on
ovarian function are difficult to assess in as much as cigarette smoke contains more
than 4,000 chemicals. Benzo(a)pyrene (BaP), an endocrine disruptor that perturbs
ovarian function, is one of the major components of cigarette smoke. The objective
of this study was to study the mechanism by which BaP perturbs normal functioning
of the ovary. Female F-344 rats with regular four-day estrous cycles were used in
this study. Rats were administered BaP (5mg BaP/kg) or vehicle (tricaprylin; control)
by oral gavage for 30 days. A week after the onset of gavaging, daily vaginal
smears were conducted to determine the ability of BaP-exposed rat to cycle continuously.
At the end of the treatment regimen, blood samples were collected from rats
at proestrus for the determination of serum estradiol-17β (E 2 ), LH and FSH concentrations.
Subsequently, ovaries were harvested post CO 2 asphyxiation for determining
the expression of pro-inflammatory biomarkers. BaP significantly increased
estrous cycle length by approximately 3 days. Serum concentrations of E 2 and LH
were decreased (P