The Toxicologist - Society of Toxicology

males per group were dosed orally once daily from PND 22 to 42/43. The five
groups in Set A were dosed with 0 mg/kg/day in corn oil, 25 or 100 mg/kg/day
CN, and 30 or 60 mg/kg/day DE 71. The three groups in Set B were dosed with 0
mg/kg/day in corn oil, and 12.5 or 50 mg/kg/day MET. Animals were examined
for age and body weight at vaginal opening [VO] daily, beginning on PND 22; day
of first estrous (DFE) and vaginal cytology. Necropsies were performed 2 hours
post-dose on PND 42/43. Selected organs were weighed; ovaries, pituitary glands
and thyroids were fixed for histological examination; and serum was collected for
hormone analysis (thyroxine [T4] and thyroid-stimulating hormone [TSH]). There
were no clinical signs of toxicity or effects on feed consumption during the study.
There was a decrease in body weight and/or gain associated with CN and MET.
Endocrine disruptive effects of: CN at 25 and/or 100 mg/kg/day included reduced
organ weights of the ovaries and pituitary gland, a marked delay in VO and in the
DFE, altered hypertrophic/hyperplasic uterine scores; decreased T4 and increased
TSH. DE-71 at 30 and/or 60 mg/kg/day included reduced ovarian weight, increased
thyroid weight; decreased thyroid colloid areas scores; decreased T4 and increased
TSH; and delayed VO and DFE. MET at 12.5 and/or 50 mg/kg/day included
reduced ovarian weight, advanced VO, irregular vaginal cytology (extended
estrous). Conclusion: The applied experimental design satisfactorily detected the
endocrine disruptive potential of the xenobiotics used in this validation.
2111 MALE PUBERTAL ASSAY: VALIDATION USING KNOWN
ENDOCRINE DISRUPTORS.
R. M. Parker 1 , E. R. Frizell 1 , E. Horsley 2 and K. Hazelden 3 . 1 DART, Huntingdon
Life Sciences, East Millstone, NJ, 2 Pfizer, Sandwich, United Kingdom and
3
MedImmune, Cambridge, United Kingdom.
This study was performed to validate the ability of this assay to detect the endocrine
disruptive potential of xenobiotics Vinclozolin (VN), DE 71, 1-Chloro-2-
Nitrobenzene (CN) and Dibutyl Phthalate (DP) in pubertal male rats. Fourteen
(Set A) and 15 (Set B) males per group were dosed orally once daily from PND 23
to 52/53. The 5 groups in Set A were dosed with 0 mg/kg/day in corn oil, 30 or
100 mg/kg/day VN, and 30 or 60 mg/kg/day DE 71. The 5 groups in Set B were
dosed with 0 mg/kg/day in corn oil, 25 or 100 mg/kg/day CN, and 500 or 1000
mg/kg/day DP. Rats were examined for preputial separation [PS] daily from PND
30. Necropsies were done 2 hours post-dose on PND 52/53. Selected organs were
weighed; testes, epididymides and thyroids were histologically evaluated; and serum
was analyzed for hormones (thyroxine [T4], thyroid-stimulating hormone [TSH],
and testosterone [T]). There were no clinical signs of toxicity or effects on feed consumption.
There was a slight decrease in body weight gain from PND 23 to 52/53
associated with DE 71 and VN. Endocrine disruptive effects of: DP at 500 and/or
1000 mg/kg/day included decreased size and organ weights for the testis, epididymis
and seminal vesicles (SV); reduced levator ani/bulbocavernosus [LABC]
muscle weight; severe seminiferous tubule atrophy with totally depleted germ cells;
reduced T4, TSH and T levels; and a delay in PS. VN at 30 and/or 100 mg/kg/day
included reduced epididymis, SV, and LABC muscle and increased testis weights;
reduced T4 and increased T levels; and a delay in PS. CN at 100 mg/kg/day included
reduced ventral prostate, SV, LABC muscle; reduced TSH and increased T
levels; and a delay in PS. DE 71 at 30 and/or 60 mg/kg/day included reduced
LABC muscle and increased testis weights; reduced T4 and T and increased TSH
levels; increased thyroid epithelial cell height, decreased colloid area; and a slightly
delayed PS. Conclusion: The applied experimental design satisfactorily detected the
endocrine disruptive potential of the xenobiotics used in this validation.
2112 INTERACTION OF PERFLUOROALKYL ACIDS WITH
HUMAN ESTROGEN RECEPTOR ALPHA.
A. D. Benninghoff, D. Koch, W. Bisson, S. K. Kolluri and D. E. Williams.
Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR.
Previously, we reported that multiple perfluoroalkyl acids (PFAAs) bind to the estrogen
receptor (ER), induce expression of the estrogen biomarker protein vitellogenin
and enhance liver carcinogenesis in rainbow trout, an animal model that
represents the lack of sensitivity to peroxisome proliferators observed in humans.
We now report new results from two studies examining the interaction of several
PFAAs with human ERα. First, human embryonic kidney cells were transfected
with the minimal SV40 promoter driving a luciferase gene containing the estrogen
responsive element (ERE) and human ERα. Cells were then treated with estradiol
(E2), perfluorooctanoic (PFOA), -nonanoic (PFNA), -decanoic (PFDA) and undecanoic
(PFUnDA) acids as well as perfluorooctane sulfonate (PFOS) and 8:2 fluorotelomer
alcohol (FtOH). PFOA, PFNA, PFDA and PFOS all significantly induced
gene reporter activity up to 2.5-fold at concentrations ranging from 10 to
1000 nM (PFOS increased reporter activity at 1 nM). PFUnDA and FtOH did not
markedly alter gene reporter activity. Second, we employed a computational model
using an iterative scoring method based upon 3D coordinates of the human ERα
ligand binding domain complexed with E2 to investigate molecular docking of
these putative ligands with the receptor. PFOA, PFNA, PFDA and PFOS all efficiently
docked with hERα and formed a hydrogen bond at residue Arg394, similar
to other known xenogestrogens bisphenol A and nonylphenol. Interestingly, FtOH
was found to effectively dock in this model similarly to the native hormone E2,
which formed hydrogen bonds with both the Arg394 and Glu353 residues.
PFUnDA did not interact with ERα in this computational model. These data support
the contention that several PFAAs may be xenoestrogens. Supported by NIH
grants ES07060, ES03850 and ES00210.
2113 THE ATRAZINE METABOLITE DACT CAUSES
SUPPRESSION OF LH RELEASE IN LβT2 PITUITARY
CELLS.
A. C. Schell, R. B. Tjalkens, M. E. Legare and W. Hanneman. Colorado State
University, Fort Collins, CO.
Chlorotriazine (Cl-TRI) compounds such as atrazine (ATRA) are among the most
commonly used herbicides worldwide. Exposure to ATRA and other Cl-TRIs
causes a spectrum of reproductive and developmental deficits in laboratory species
that involves selective disruption of the hypothalamic-pituitary gonadal (HPG) axis
and blunting of the surge of leutinizing hormone (LH) from the pituitary gland induced
by gonadotropin releasing hormone (GnRH). While this effect is well
known, the mechanism of action by which the chlorotriazines blunt the LH surge
is not known. Calcium is known to play a central role in the secretion of LH from
the pituitary gonadotrophs. Therefore, the objective of the present study was to elucidate
if exposure to the atrazine metabolite diaminochlorotriazine (DACT) alters
GnRH-induced intracellular Ca2+ release. LβT2 cells were treated for 24 hrs with
300μM DACT and imaged for intracellular calcium utilizing the fluorescent probe
Fluo-4 AM. Images were captured every 2 sec and 10nM GnRH was added at 30
sec to stimulate intracellular Ca2+ release. Subsequent images were collected for 5
minutes. DACT exposure resulted in a reduction in the GnRH-induced cytosolic
Ca2+ transient. This decreased Ca2+ response correlated with loss of Ca2+ from
thapsigargin-sensitive ER stores but could be recovered using the Ca2+ ionophore,
A23187. Secretion of LH was also significantly reduced in DACT treated cells and
was partially restored by treatment with A23187. These results suggest that DACT
directly targets G protein-coupled Ca2+ release signals in LβT2 cells to inhibit
GnRH-stimulated release of LH.
This work was supported by a grant from the College of Veterinary Medicine
2114 A 28-DAY TOXICITY STUDY IN JUVENILE BEAGLE
DOGS WITH A ONE MONTH RECOVERY FOLLOWING
ORAL ADMINISTRATION OF FAROPENEM
MEDOXOMIL.
A. S. Faqi 1 , C. Lanphear 1 , S. Gill 2 and D. B. Colagiovanni 2 . 1 Developmental &
Reproductive Toxicology, MPI Research, Mattawan, MI and 2 Toxicology, Replidyne,
Inc., Louisville, CO.
Faropenem Medoxomil (FM) is an orally active beta-lactam antibiotic belonging to
the penem group which is being developed for community antibiotic use. The objective
of this study was to determine the toxicity of FM when administered to
neonatal/juvenile dogs for 28 consecutive days. The study consisted of a vehicle
control and four treatment groups of five male and female dogs /group (2-3 weeks
of age at study initiation). The treatment groups received FM at dose levels of 100,
300, 600, or 1400 mg/kg/day (25, 75, 150, or 350 mg/kg/dose, respectively). FM
or vehicle was administered to all groups via oral gavage, four times a day at a dose
volume of 5 mL/kg/dose. Blood samples for clinical pathology evaluations were collected
from all animals at pretest, during Week 2, and prior to the terminal (Week
4) and recovery necropsies. Urine samples were collected from all animals prior to
terminal and recovery necropsies via cystocentesis. TK samples were collected from
all animals at designated time points on Days 1 and 27. Following 28 days of administration,
two animals /sex at 0 and 1400 mg/kg/day were maintained for a 1
month recovery period and then sacrificed with full necropsy. Body weight was significantly
decreased in male puppies at 1400 mg/kg/day when compared to the
control male animals. Comparison of Day 27 with Day 1 TK parameters showed a
change in faropenem pharmacokinetic behavior over time with an apparent increase
in the rate of clearance as observed in decrease in AUC0-6 with little to no
change in Cmax and a decrease in Tmax values. Kidney lesions consisting of minimal
to mild vacuolation and tubular degeneration/necrosis were observed at 1400
mg/kg/day. Based on these results, the No Observed Adverse Effect Level
(NOAEL) for general toxicity was 600 mg/kg/day based on kidney vacuolization
and tubular degeneration/necrosis observed at 1400 mg/kg/day.
SOT 2010 ANNUAL MEETING 449