The Toxicologist - Society of Toxicology

the antral follicles was monitored every 24h for a period of 96h. Further, we examined
the gene expression levels of enzymes metabolizing MXC Cyp1a2, Cyp2c29
and Cyp3a4 in the liver of ESR1 OE and control mice. Cyp1a2 and Cyp2c29 metabolize
MXC, while Cyp3a4 metabolizes MOH and HPTE to their subsequent
metabolites. The data show that at 96h, the cultured antral follicles from ESR1 OE
antral follicles are more susceptible to toxicity induced by MXC, MOH and HPTE
because low doses of these chemicals caused follicle growth inhibition in ESR1 OE
mice, but not in control mice. In the livers, our results showed that while there was
no difference in the mRNA levels of Cyp1a2 and Cyp2c29 between ESR1 OE and
control livers, the mRNA levels of Cyp3a4 were significantly lower in ESR1 OE livers
compared to controls (ESR1 OE fold-change over control = 1: Cyp1a2 = 1.2;
Cyp2c29 = 1.1; *Cyp3a4 = 0.4; n = 5; *p ≤ 0.05). Collectively, these data suggest
that ESR1 OE mice may be more susceptible to toxicity induced by MXC and its
metabolites because of low clearance of the metabolites by the liver. Support: NIH
R21ES13061, R01ES012893 and an Eli Lilly Fellowship in Toxicology.
1039 MONO- AND DI-ESTER PHTHALATES ALTER
TESTOSTERONE PRODUCTION IN MOUSE BLTK1
MURINE LEYDIG TUMOR CELLS.
Q. Ding 1 , N. A. Rahman 2 , I. T. Huhtaniemi 2 and T. R. Zacharewski 1 .
1 Department of Biochemistry & Molecular Biology and Center for Integrative
Toxicology, Michigan State University, East Lansing, MI and 2 Department of
Physiology, University of Turku, Turku, Finland.
Phthalates, widely used plasticizers in a variety of consumer and industrial products,
have been implicated in the etiology of rodent testicular dysgenesis by altering
hormone levels. In this study, BLTK1 cells, a subclone of a murine Leydig cell line
isolated from a testicular tumor in a inhibinα/SV40Tag transgenic mouse, are used
to study the effects of phthalates and their monoester metabolites (i.e., DMP -
mMP, DEP - mEP, DBP - mBP, BBzP - mBzP, DHP - mHP and DEHP - mEHP)
on steroidogenesis. BLTK1 cells possess an intact steroidogenic pathway that produces
low basal levels of progesterone (P) and testosterone (T) which is inducible
by recombinant human chorionic gonadotropin (rhCG) as measured by ELISA.
RT-PCR and western blot verified the expression of StAR, Cyp11a, Hsd3b1,
Cyp17a1, Hsd17b3 in BLTK1 cells. rhCG (0.01 – 100 ng/ml) exhibited time- and
dose-dependent induction of P and T levels with EC50 values of 1.8 and 1.5
ng/ml, respectively, at 4 h. In rhCG time course studies, maximum P induction was
achieved at 4 h, which returned to basal levels by 24 h while T levels gradually increased
until 8 h and then remained relatively constant. mEHP also elicited marked
time- and dose-dependent induction of P and T levels, while DBP, BBzP, and mHP
induced moderate levels at 24 h. DMP, mMP, DEP, mEP, mBP, mBzP, DHP, and
DEHP elicited negligible effects on P and T levels. QRT-PCR results indicate that
mEHP exerts its effects by altering steroidogenic gene expression. More specifically,
mEHP induced StAR mRNA levels while repressing the expression of Cyp17a1
and Srd5a1. Overall, these results suggest that selected mono- and di-ester phthalates
alter T biosynthesis by regulating the expression of specific steroidogenic
genes. Partial funding was provided by the American Chemistry Council.
1040 A STUDY OF FERTILITY IN SPRAGUE-DAWLEY RATS
WITH XOMA 052, A NOVEL MONOCLONAL
ANTIBODY TARGETING IL-1 BETA.
C. Gasper 1 , B. Thorsrud 2 , J. Ma 1 , L. Cao 1 , K. Der 1 and K. Meyer 1 . 1 Product
Development, XOMA (U.S. ) LLC, Berkeley, CA and 2 Developmental & Reproductive
Toxicology, MPI Research, Mattawan, MI.
XOMA 052 is an ultra-high affinity (300 fM) humanized IgG2 monoclonal antibody
that specifically binds to IL-1 beta and inhibits activation of the IL-1 receptor.
This activity is expected to prevent the cellular signaling events that produce inflammation
associated with the development of diseases such as Type 2 diabetes,
rheumatoid arthritis and Behçet’s disease. The rat was selected for this fertility study
due to similar binding affinity and in vitro functional activity between human and
rat IL-1 beta with XOMA 052. Male and female Sprague Dawley rats were administered
XOMA 052 or vehicle by subcutaneous injection once weekly at dose levels
of 0, 3, 30, and 90 mg/kg. Male rats were dosed weekly starting 28 days prior to
pairing and continuing through euthanasia. Female rats were dosed weekly starting
14 days prior to pairing until positive evidence of copulation was observed and
again on GD 4. Mated females were each euthanized on GD 13 and all males were
euthanized upon competition of the final GD 13 uterine examination.
Observations and evaluations included clinical signs, gestation body weights and
body weight change, food consumption, immunogenicity, macroscopic pathology,
and reproductive performance (estrous cyclicity, reproductive and fertility indices,
uterine and ovarian examinations, and sperm evaluation). All animals survived to
scheduled necropsy. Results showed no toxicologically significant differences in any
of the parameters evaluated. Based on the results, the no-observed-adverse-effect
222 SOT 2011 ANNUAL MEETING
level (NOAEL) for general and reproductive toxicity for male and female rats was
considered to be 90 mg/kg, the highest dose level tested. In conclusion, XOMA
052 was well tolerated and did not affect male or female fertility parameters or have
any untoward effects during the period of early embryonic development to implantation
in the rat.
1041 IMMUNOHISTOCHEMICAL AND CHIP MICROARRAY
ANALYSIS OF PPARα IN FETAL RAT TESTES EXPOSED
TO DIBUTYLPHTHALATE (DBP).
S. M. Plummer 1 , D. Dan 1 , J. Quinney 1 , M. Millar 3 , M. Sheila 3 , N. Hallmark 2 ,
R. D. Phillips 2 and C. R. Elcombe 1 . 1 CXR Biosciences, Dundee, United Kingdom,
2 ExxonMobil Petroleum & Chemical, Hermeslaan, Belgium and 3 MRC Human
Reproductive Sciences Unit, Edinburgh, United Kingdom.
Previous “ChIP on chip” studies indicated an increase in binding of peroxisome
proliferator receptor alpha (PPARα) to the Cyp11a promoter in gestational day 15
(GD15) rat testes could account for reduced Cyp 11a expression following DBP exposure
(Plummer et al (2010) The Toxicologist CD, 114:1488). It was postulated
that PPARα hinders transcription factor (SF-1) binding to the Cyp11a promoter,
restricting Cyp11a expression and reducing testicular steroidogenesis. To test this
hypothesis (1) ChIP microarray data analysis (GD15 and GD19 testes) on additional
SF-1-regulated steroidogenic genes and (2) immunohistochemical staining
(GD15 testes) for PPARα were conducted. ChIP microarray was performed (see
ref. above) on GD15 and 19 fetal testes from Wistar rats exposed in utero to DBP
(500mg/kg, p.o. to dams from GD12). Bouin’s fixed GD15 fetal testes were immunostained
using antibodies against PPARα and 3βHSD, a Leydig cell marker.
ChIP array data showed DBP increased PPARα binding in StAR and Cyp17a promoter
regions in GD15 testes and inhibited SF1-binding in StAR and Cyp17a promoter
regions in GD19 testes correlating with reduced expression of these SF-1regulated
steroidogenic genes. Immunostaining results (GD 15) confirmed PPARα
protein expression in interstitial regions of the testes partly colocalised with
steroidogenic protein 3βHSD in Leydig cells. These data are consistent with expression
microarray data (Plummer et al (2007) Toxicol Sci 97:520) and suggest
that DBP-induced alterations in PPARα binding could interfere with SF-1 binding
in steroidogenic gene promoters. As PPARα is expressed primarily in immature
Leydig cells it is likely that interference with SF-1 binding/ transactivation precedes
functional maturity in target cells (3βHSD). The data are consistent with the hypothesis
that PPARα could in part mediate DBP’s anti-androgenic effects. This
work was supported by ExxonMobil Petroleum & Chemical
1042 GESTATIONAL EXPOSURE TO ATRAZINE HAS LITTLE
EFFECT ON FEMALE REPRODUCTIVE PARAMETERS
IN SPRAGUE-DAWLEY (SD) RATS.
L. K. Davis, A.S.Murr, T. E. Stoker, M. Narotsky, J. M. Goldman and R. L.
Cooper. Endocrine Toxicology Branch, NHEERL, U.S. EPA, Research triangle pk, NC.
Atrazine (ATR), a commonly used herbicide, has been shown to exert reproductive
effects in animals; however, most of these studies have focused on adult exposure.
In contrast, there is limited information available on the reproductive and developmental
effects of gestational exposure to this herbicide. Here, we examined the effects
of 0, 1, 5, 20 and 100 mg/kg ATR administered to SD dams on gestational
days 14-21, a key developmental window, on female reproductive end points.
Dosing occurred once daily or was divided into two doses per day; the lower doses
were chosen based on existing female LOELs, while 100 mg/kg served as a positive
control. 100 mg/kg decreased both maternal and offspring body weights, and postnatal
mortality was higher in this group shortly after birth. By postnatal day (PND)
21 no differences in offspring body weights were present. Age of vaginal opening,
used as a pubertal index, was significantly delayed only in the 100 mg/kg group. No
differences in body weights were seen at this time. Based on recently established
guidelines, mammary gland whole mounts were prepared on PND 45 and analyzed
by individuals blind to experimental treatment. There were no apparent treatment
effects on mammary gland development, including the number of terminal end
buds and branch points. To determine the effects of gestational ATR exposure on
estrous cycles in the adult offspring, vaginal smears were examined until the control
animals reached reproductive senescence (approximately 9 months). These tracking
data indicated that animals in the highest treatment group continued to cycle over
this time, while growing numbers of females in the lower dose groups showed an
emerging acyclicity. In brief, these data indicate that gestational exposure to ATR at
doses as high as 100 mg/kg in the dam, had few adverse effects on these standard
measures of reproductive development. This is an abstract of a proposed presentation
and does not necessarily reflect EPA policy.