The Toxicologist - Society of Toxicology

2372 EFFECT OF NOREPINEPHRINE, ENDOGENOUS
DOPAMINE, AND EXOGENOUS DOPAMINE ON
PROLACTIN RELEASE IN AN OVARIECTOMIZED
RAT MODEL.
D. A. Brott 1 , P. Campbell 1 , P. Bentley 1 , H. Andersson 2 , J. Stewart 3 , R. Huby 3
and L. Kinter 1 . 1 Global Safety Assessment, R&D, AstraZeneca Pharmaceuticals,
Wilmington, DE, 2 Global Safety Assessment, R&D, AstraZeneca Pharmaceuticals,
Södertälje, Sweden and 3 Global Safety Assessment, R&D, AstraZeneca
Pharmaceuticals, Alderley Park, United Kingdom.
Dopaminergics (D) can produce unwanted endocrine side-effects in rats; D2 receptor
agonists inhibit prolactin release. To characterize the mechanism, this study
evaluated effects of central-acting: 1) exogenous dopamine (dopamine agonist,
bromocriptine), 2) endogenous dopamine (dopamine transporter, DAT inhibitors,
mazindol and GBR12909) and 3) endogenous norepinephrine (NE, NE transport
inhibitor, nisoxetine), and 4) peripheral-acting exogenous dopamine (dopamine agonist,
carmoxirole) on prolactin release using double cannulated ovariectomized
rats. Rats were dosed with estradiol (E2, 0.6 - 20 ug/rat, iv), blood collected every
30 minutes between 1.5 to 5 hours, and evaluated for prolactin and luteinizing hormone
(LH). E2 at 2 ug/rat (iv) was deemed the optimal concentration based on
maximum prolactin release and expected suppression of LH. In remaining experiments,
rats were dosed with E2 (2 ug/rat, iv) one hour prior to compound treatment
(iv), blood collected 0.5 to 4 hours, and evaluated for prolactin. As expected
the D2 receptor agonist bromocriptine (1 mg/kg) inhibited the E2-induced prolactin
release, and so did the DAT inhibitors mazindol (5 mg/kg) and GBR12909
(3 mg/kg). Carmoxirole (15 mg/kg) induced a rapid initial release of prolactin, and
inhibited E2-induced release. E2-induced prolactin release was not inhibited by the
central acting NE uptake inhibitor nisoxetine (10 mg/kg). This study qualified the
double cannulated ovariectomized rat model for evaluating the impact of central
and peripheral acting compounds, and suggests that the mechanism may be activated
both inside and outside the blood-brain barrier with D but not NE.
2373 IDENTIFICATION OF ENDOCRINE DISRUPTORS
USING AN ORGANOTYPIC VAGINAL TISSUE MODEL.
S. Ayehunie, K. LaRosa, T. Landry, J. E. Sheasgreen and M. Klausner. MatTek
Corp, Ashland, MA. Sponsor: P. Hayden.
Environmental or occupational exposure to a broad variety of chemical agents
can alter normal endocrine function. The effects of these “endocrine disruptors” (ED)
can have serious health implications including deleterious effects to reproductive
capacity, fetal development, the immune system, and carcinogenesis. Current animal
tests are expensive, use a large number of animals, and are not necessarily applicable
to humans. In addition, the Cosmetics Directive bans the use of animals
for studies involving a broad variety of cosmetic and personal care products. In this
study, we investigated the potential use of an organotypic tissue model,
EpiVaginal TM , for Tier 1 screening of chemicals that may be agonists or antagonists
of the estrogen receptor (ER). MTT, H&E staining, RT-PCR, and ELISA assays
were used to define tissue viability, structure, gene expression, and estrone release
patterns, respectively. Based on the MTT studies, only concentrations resulting in
>50% tissue viability were used. H&E stained tissue cross-sections showed thinning
of the basal and parabasal layers following 72 hour exposure to ER antagonists
when compared to the control tissues; exposure to ER agonists resulted in thicker
basal and parabasal layers, indicating stimulation of cellular proliferation. RT PCR
analysis showed an increase in progesterone receptor B (PRb) levels for 3 of 3 agonists
and a decrease or no change for 6 of 8 antagonists when compared to negative
controls. Furthermore, Er-α expression increased following exposure to the ER agonists
and decreased following exposure to ER antagonists. ELISA assays showed
increased estrone release by ED agonists but not ED antagonists. Based on estrone
release (n=22 test articles), a prediction model (PM) for ER agonists was established.
The PM identifies ER agonists with a high sensitivity (85.7%), specificity
(100%), and accuracy (95.5%). In conclusion, the EpiVaginal tissue appears to be a
useful in vitro model to screen for chemicals with endocrine disrupting potential.
2374 EVALUATION OF SUBCHRONIC TOXICITY AND
ESTROGENIC ACTIVITY OF BLACK COHOSH IN
FEMALE WEANLING B6C4F1 MICE EXPOSED BY
GAVAGE.
M. Mercado-Feliciano 1 ,M. D. Stout 1 ,C. A. Granville 2 ,M. Hejtmancik 2 ,R.
Newbold 1 ,M. K. Vallant 1 and P. M. Foster 1 . 1 National Toxicology Program, NIEHS,
Research Triangle Park, NC and 2 Battelle Memorial Institute, Columbus, OH.
Black cohosh (Actaea racemosa) is a popular herbal supplement for the treatment of
gynecological symptoms. The recommended dose is 40 mg/day (~0.5–0.6
mg/kg/day). The current scientific literature suggests black cohosh extract may re-
510 SOT 2011 ANNUAL MEETING
duce luteinizing hormone secretion in ovariectomized rats, but demonstrations of
estrogenic activity remain inconclusive. The National Toxicology Program (NTP) is
currently assessing the possible toxicity and estrogenic effects of black cohosh in in
vivo rodent models. We previously reported that a 90-day NTP study showed a 3day
delay in vaginal patency in Wistar Han rats, suggesting the possibility of antiestrogenic
activity. In addition, the thymus was the only major target organ in the
rat study. However, no significant estrogenic effects were observed in an immature
CD-1 mouse uterotrophic assay at up to 100 mg/kg/day delivered by subcutaneous
injection (higher doses caused acute toxicity). A more recent NTP 90-day study
was conducted in weanling female B6C3F1 mice administered an ethanolic extract
by gavage (in 0.5% aqueous methyl cellulose) of 0, 62.5, 125, 250, 500 or 1000
mg/kg/d black cohosh. As in the previous rat study, time to vaginal patency and
analysis of estrous cyclicity were included in addition to the routine toxicity endpoints.
There was no apparent in-life toxicity following exposure. A dose related increase
in platelets was statistically significant only for the 250 and 1000 mg/kg
groups, similar to that seen in the rat. Increased liver weights were observed at 500
mg/kg and 1000 mg/kg, a trend very similar to that observed previously in rats. A
2-day delay in vaginal patency at 125, 250 and 1000 mg/kg was not statistically significant.
With the exception of increased liver weight, we observed no other systemic
effects and no evidence of estrogenic activity in mice.
2375 IMPLEMENTATION OF TIER I MAMMALIAN ASSAYS
FOR THE U.S. EPA ENDOCRINE DISRUPTOR
SCREENING PROGRAM (EDSP).
S. Papineni 1 , A. Tobia 2 and M. S. Marty 3 . 1 Dow AgroSciences, Indianapolis, IN,
2 Nufarm Americas, Inc., Cary, NC and 3 The Dow Chemical Company, Midland, MI.
Tier I screening under the EPA EDSP has begun for compounds on the first priority
list. For the mammalian Tier I EDSP screens, dose level setting is a critical variable
that can affect assay outcome. However, dose selection is challenging as each
assay uses animals of different ages that are intact, peripubescent or castrated.
Furthermore, there are little or no previous data on gavage dosing in animals at
these young ages and the definition of maximum tolerated dose (MTD) varies by
assay. One approach is to conduct a two-week probe study via gavage in which male
and female rats (4-5/dose) are exposed to test compound from PND 22-36 (females)
or 30-44 (males), then clinical observations, body weight, clinical pathology
parameters and organ weights are examined. Histopathology may be included depending
on the compound and its target organ. These data, coupled with previous
toxicity data from the OSRI document (if available), can be used to select dose levels
for Tier I assays, potentially using fewer animals than other approaches. Once
doses are selected, use of MTD doses may raise issues with assay specificity. For example,
the Hershberger assay is designed to detect compounds that interact with
the androgen receptor (AR) or inhibit 5-alpha-reductase; however, positive results
for anti-androgenicity can be obtained with compounds that enhance steroid hormone
clearance. For the pubertal assays, caution is warranted in interpreting assay
results in the presence of significant decreases in terminal body weight. The pubertal
assay test guidelines also provide performance criteria that may not be met by
some laboratories; the three laboratories in the inter-laboratory validation only partially
met these criteria. Thus, when implementing the Tier I mammalian assays,
dose selection and performance criteria remain a challenge. However, using both
EDSP screening data and previous toxicity data in a “weight of evidence” evaluation
will provide the most accurate evaluation of potential endocrine activity.
2376 EFFECTS OF FASTING ON ENDOGENOUS PTH
LEVELS IN CYNOMOLGUS MONKEYS.
C. Ruh 1 , N. Doyle 1 , P. Bednarek 2 and S. Y. Smith 1 . 1 Charles River Laboratories,
Senneville, QC, Canada and 2 Cytochroma Inc., Markham, ON, Canada.
PTH is an important endocrine regulator of calcium and phosphorus and is routinely
assessed in studies affecting bone metabolism. The objective of this study was
to determine the effects of fasting on endogenous PTH levels in cynomolgus monkeys.
Blood samples were taken from 6 monkeys per sex, 1.5 to 3.5 years old, on 4
occasions under fasted or non-fasted conditions and serum PTH levels compared.
Samples were analyzed using an ELISA kit (catalogue No. 60-3100, Immutopics).
Animals had access to certified commercial primate food twice daily. Blood was
sampled in the late afternoon at approximately the same time of day on each occasion
with or without a 7-hour pre sampling fasting period. The first set of samples
was collected under fasted conditions (Day 1); the second and third under nonfasted
conditions (Day 8 and Week 6); and the last sample was collected under
fasted conditions (Week 7). Day 1 results were quantifiable for all animals with
means of 56.78 pg/mL and 34.20 pg/mL for males and females, respectively. Day 8
results were below the lower level of quantitation (LLOQ = 13.2 pg/mL) for 4/6
males with a mean of 23.15 pg/mL for the remaining 2 individuals, while 4/6 females
had results below LLOQ, with a mean of 23.55 pg/mL for the remaining 2