The Toxicologist - Society of Toxicology

1043 THE EFFECTS OF ENDOCRINE DISRUPTION ON THE
DEVELOPING HUMAN FETAL PROSTATE.
C. Saffarini, S. J. Hall and K. Boekelheide. Brown University, Providence, RI.
Prostate cancer is the most commonly diagnosed cancer and is the second highest
cancer-related cause of death in men. This high incidence has led to speculation
about the fetal origins of this adult disease and its etiology. Exposure to exogenous
estrogens in pregnant women has been postulated to disturb the normal development
of the human fetal prostate by disrupting the natural hormonal balance.
Previous studies demonstrate that exposure to estrogens can cause some degree of
epithelial and stromal hyperplasia, inflammation, and prostatic intraepithelial neoplasia
(PIN) lesions. The present study used a xenograft model to characterize and
determine the differentiation of the human fetal prostate implants (gestation 12-22
weeks) exposed to either corn oil (control) or 250 ug/kg/body weight of 17β-estradiol
3-benzoate during the first few days post-transplant. This xenograft model uses
the renal subcapsular space as the site of implantation, thereby allowing for proper
vascularization and growth of the implant. Immunohistochemical (IHC) characterization
included markers specific for stromal (desmin, smooth muscle alpha actin,
myosin, vinculin, vimentin) and epithelial maturation (cytokeratins, lamin A/C, ecadherin,
p63, prostate specific antigen), neuroendocrine cells (chromogranin A),
and hormone receptors (androgen receptors, estrogen receptor alpha and beta). In
addition, proliferation as well as apoptosis was noted using KI-67 and TUNEL
staining. As expected, the prostate implants appear to grow and mature as seen in
the 7, 14 and 30 day time points following xenografting. Interestingly, preliminary
IHC results reveal a decrease in both KI-67 and e-cadherin expression, as well as an
increase in PSA expression in the 7-day estradiol treated implants. This unique
xenograft model will provide insight on the growth and development of the human
fetal prostate following developmental endocrine disruption.
1044 TWO-GENERATION REPRODUCTIVE TOXICITY
STUDY OF ALUMINIUM SULFATE ADMINISTERED
VIA DRINKING WATER TO RATS.
M. Hirata-Koizumi 1 , S. Fujii 2 , A. Ono 1 , A. Hirose 1 , R. Hasegawa 1 , T. Imai 3 ,
K. Ogawa 1 , M. Ema 1 and A. Nishikawa 1 . 1 National Institute of Health Sciences,
Tokyo, Japan, 2 Safety Research Institute for Chemical Compounds Co., Ltd., Sapporo,
Japan and 3 Central Animal Laboratory, National Cancer Center Research Institute,
Tokyo, Japan.
In order to evaluate the human health risk by intake of aluminium from food and
drinking water, the 67th JECFA meeting clearly stated the need for a multigenerational
study on a relevant aluminium compound. We conducted a two-generation
reproductive toxicity study (OECD GD416) in SD rats given drinking water containing
aluminium sulfate at 0, 120, 600 or 3000 ppm. In both F0 and F1 generations,
water consumption was decreased in males and females of all treatment
groups. Decreased food consumption was observed in both sexes of F0 and F1 animals
in the 600 and 3000 ppm groups, and the body weight in F0 males and females
was decreased only at 3000 ppm. In either generation, no compound-related
changes were found in the reproductive parameters. While there were also no
changes in birth weight of F1 and F2 pups, decreased body weights on postnatal
day 21 were observed in male and female F1 pups and female F2 pups in the 3000
ppm group. Although there were decreases in the liver and spleen weight in both
sexes of F1 and F2 weanlings at 3000 ppm, no histopathological changes were detected
in these organs. Vaginal opening was slightly delayed in F1 females at 3000
ppm, but there was no significant difference in the body weight at the time of attainment
between the control and 3000 ppm groups. There were no compound-related
changes in other developmental parameters, including behavioral parameters.
In conclusion, the NOAEL for the reproductive/developmental toxicity was considered
to be 600 ppm, which was corresponding to the total aluminum intake of
8.06 mg Al/kg bw/day in rats by considering aluminium content in the standard
diet (25-29 ppm).
1045 APPLICATION OF IN VIVO AND IN VITRO TOOLS FOR
DETECTION OF EMBRYOTOXICITY: THE EXAMPLE
OF ARTEMISININ-TYPE COMPOUNDS.
P. Colombo, M. Longo, S. Zanoncelli and M. Brughera. Preclinical Development,
Accelera Srl, Nerviano - Milano, Italy.
The investigation of embryotoxicity using conventional in vivo experimental designs
(e.g. ICH S5A), can be enhanced when combined with a panel of complementary
in vitro embryotoxicity tests, namely the rat whole embryo culture, micromass,
frog embryo and zebrafish embryo teratogenesis assays. Here we present a
case in which a combination of in vivo and in vitro embryotoxicity assays were applied
to the study of the anti-malarial drug dihydroartemisinin, demonstrating the
potential use of in vitro data during drug discovery as an early stage indicator of potential
liabilities. Artemisinin derivatives are a cornerstone of contemporary malaria
treatment, although embryotoxicity remains a critical issue, as infection with
Plasmodium falciparum is dangerous to both mother and child during the first
trimester of pregnancy. Initial conventional in vivo studies indicated that
artemisinin-type compounds were embryolethal in experimental animals through
an unknown mechanism. Subsequent ad hoc in vivo studies in the rat helped identify
a narrow window of susceptibility of the embryo to artemisinins.
Complimentary in vitro assays were then able to identify a species-nonspecific
mechanism of embryotoxicity: the depletion of primitive metabolically active
RBCs (Red Blood Cells) clonally released over a short period of time from primitive
sites of haematopoiesis. In vitro assays further helped in establishing differences in
the length of the period of susceptibility vs. the period of exposure (treatment duration)
helping the evaluation of the clinical relevance of this finding. Data obtained
using in vitro models can also provide useful feedback to drug discovery, allowing
ranking of novel potential compounds according to embryotoxicity liability. In an
attempt to establish whether and to what extent activity as anti-malarials and embryotoxicity
can be divorced, IC50s for activity in P. falciparum strains and the
NOELs for RBCs were compared for various compounds, demonstrating that in
certain cases a good safety margin can exist.
1046 DEVELOPMENT OF REPRODUCTIVE ORGANS IN
MALE CYNOMOLGUS MONKEYS.
E. Haruyama 1, 2 , Y. Ayukawa 1 , K. Kamura 1 , M. Mizutamari 1 , Y. Ooshima 1 , T.
Sukamoto 1 , R. Nagata 1 and A. Tanimoto 2 . 1 Shin Nippon Biomedical Laboratories,
Ltd., Kagoshima, Kagoshima, Japan and 2 Kagoshima University, Kagoshima,
Kagoshima, Japan.
Purpose: We examined reproductive organs in control group cynomolgus monkeys
(136 males) used in toxicity studies from August 2008 to August 2009 to evaluate
their development histopathologically and morphometrically. Result: A clear correlation
was noted between histopathological degree of testicular maturity, as classified
into six grades (1: Immature, 2: Pre-pubertal, 3: Onset of puberty, 4: Pubertal,
5: Early adult, 6: Adult) and testis weight; however, large individual differences in
the relationships with age and body weight were recognized. Increases in the epididymis
and seminal vesicle weights accompanying increased testis weight were indicated.
The prostate showed an increase in weight accompanying increased testis
weight; however, there was a tendency to remain constant at Grades 4 to 6. When
compared by the degree of testicular maturity in morphological examinations, the
areas of the ductus epididymis and the cavity of the ductus epididymis showed increases
with grade, and were notably increased at Grade 6. The proportion of the
epithelial cell layer of the seminal vesicles also showed an increase with grade. In the
prostate, glandular cavities were generally not observed with Grade 1; however, they
were visible from Grade 2, and increases in relative glandular cavity area were noted
up to Grade 4. Conclusion: In these results, a clear correlation between testis weight
and histopathological degree of maturity was noted; however, there were large individual
differences in the relationships with age and body weight, and it is considered
necessary to select animals for toxicity studies in which testicular toxicity it to
be evaluated. The prostate does develop in a manner accompanying the stage of testicular
development; however, maturity is reached at Grade 4. Conversely, the epididymides
and seminal vesicles develop in accordance with the degree of testicular
maturity, and differences in development applying to each organ were noted.
1047 INTERACTIONS OF ENDOCRINE DISRUPTING
CHEMICALS WITH THE BCRP TRANSPORTER:
POTENTIAL MECHANISM FOR DICTATING FETAL
EXPOSURE.
L. Aleksunes and X. Wen. Pharmacology & Toxicology, Rutgers University,
Piscataway, NJ.
Efflux transporters are critical determinants of environmental chemical disposition
and susceptibility to adverse events. Because in utero exposure to chemicals that interfere
with endocrine function carries the risk of developmental perturbations, it is
important to understand how endocrine disrupting chemicals (such as bisphenol A,
genistein, zearalenone, methoxychlor) interact with placental efflux transporters
such as the breast cancer resistance protein (BCRP), multidrug resistance protein 1
(MDR1), and multidrug resistance-associated proteins 1, 2, and 5 (MRP1, 2, 5).
The purpose of this study was to identify interactions between efflux transporters
and endocrine disrupting chemicals using membrane vesicles overexpressing
human isoforms of MRP1, 2, 5, MDR1, and BCRP. Functional inhibition studies
of endocrine disrupting chemicals were conducted using fluorescent prototypical
substrates and inside-out membrane vesicles overexpressing human BCRP, MDR1,
MRP1, 2, and 5. All four endocrine disrupting chemicals markedly inhibited
BCRP transport (10-40% of control) while only bisphenol A reduced MDR1
SOT 2011 ANNUAL MEETING 223