The Toxicologist - Society of Toxicology

this approach, a chemical can be considered as an endocrine disrupter when a positive
outcome in endocrine sensitive endpoints in an apical (or relevant non-apical
in vivo)study is conclusively supported by mechanistic data i.e. the sequence of the
biochemical/cellular events that underlies the adverse effect has been described and
understood. Once a chemical has been identified as an endocrine disrupter a potency
assessment should be performed based on specificity, human relevance, dose
level, exposure duration, nature/severity of adverse effects and number of species affected.
These elements should be considered collectively in a weight of evidence approach;
together with data on human exposure, the risk that a chemical may pose to
human health can then be fully evaluated.
2097 MIXTURE EFFECTS OF THREE FLAVONOID
PHYTOCHEMICALS ON ADRENAL AND SEX
HORMONE SECRETION IN THE HUMAN
ANDRENOCORTICAL CELL LINE H295R.
A. Oskarsson 1 , Å. Ohlsson 1 , N. Cedergreen 2 and E. Ullerås 1 . 1 Biomedical Sciences
and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala,
Sweden and 2 Agricultural Sciences, University of Copenhagen, Copenhagen, Denmark.
Flavonoids are abundant in plant food and commonly recognized for their estrogenic
effects. Many of the flavonoids are known to interact with cytochrome P450
enzymes, including enzymes responsible for steroidogenesis. Genistein (GEN),
daidzein (DAI) and apigenin (API) are three flavonoids known to inhibit cortisol
secretion in vitro and GEN decreases corticosterone and testosterone levels in rats.
The human adrenocortical cell line H295R expresses all enzymes required for secretion
of adrenal and sex hormones. Herein, we have investigated the effect of the
three flavonoids and their ternary mixture on aldosterone (A), cortisol (C), testosterone
(T) and estradiol (E) secretion. H295R cells were treated with GEN, DAI
and API for 24 hours at non-cytotoxic concentrations (0-10 μM), individually and
as mixture. Hormone levels in cell culture medium were analysed by ELISA. The
flavonoids caused a dose-dependent inhibition of A, C and T, with stronger effects
exerted by GEN and DAI than by API. The mixture also affected A, C and T secretion
in a dose-dependent way, which followed either of the two prediction models
concentration addition (CA) and independent action (IA). GEN and DAI, however,
showed no effect on E secretion, while API inhibited secretion at the highest
concentration. Following mixture treatment, E secretion was inhibited at a lower
concentration of API. The results indicate that single flavonoid compounds, at no
observed effect levels, may add up to an effect on hormone secretion when combined
in a mixture. Since these flavonoids are abundant in food, mixture exposure
is highly relevant. We conclude that GEN, DAI and API affect steroid secretion following
single and mixture treatment and that CA and IA can predict the mixture
effect.
2098 THE EFFECTS OF SIMAZINE, A CHLOROTRIAZINE
HERBICIDE, ON FEMALE PUBERTAL DEVELOPMENT.
L. M. Zorrilla 1, 2 , E. K. Gibson 2 and T. E. Stoker 2 . 1 Molecular Biomedical Science,
College of Veterinary Medicine, North Carolina State University, Raleigh, NC and
2
Endocrine Toxicology Branch, Toxicity Assessment Division, NHEERL, Office of
Research and Development, U.S. Environmental Protection Agency, Research Triangle
Park, NC .
Several chlorotriazine herbicides, such as atrazine and its metabolites, have been
shown to target the neuroendocrine regulation of male and female reproductive development.
Simazine is a pre-emergence herbicide used to control broad-leaf weeds
and annual grasses on citrus, nuts and corn crops. It is the second most commonly
detected pesticide in surface and ground waters in the U.S., Europe, and Australia.
Simazine is structurally related to, and shares metabolites with, atrazine; however,
no studies have evaluated the effects of simazine on pubertal development in the female
rat. Therefore, the current study examines the effects of simazine on pubertal
development and estrous cyclicity in the female rat using the U.S. EPA’s Endocrine
Disrupter Screening Program (EDSP) Female Pubertal (Tier 1) protocol (21 day
dosing period). In the first block, Wistar rats were gavaged with 0, 12.5, 25, 50, or
100 mg/kg of simazine from postnatal day (PND) 22 to 42. In a second block, the
dosing period was extended to the first day of estrus following PND 62 and an additional
group (200 mg/kg) was included. Vaginal opening was delayed at 25
mg/kg and higher and the day of first estrous was significantly delayed at 100
mg/kg and above. However, no treatment effect on the number of estrous cycles
was observed. Serum prolactin (PRL) levels were decreased following simazine exposure
at 50 mg/kg and higher. These data demonstrate that simazine delays the
onset of puberty in the female rat and decreases serum PRL similar to other chlorotriazines.
Extension of the dosing period enables monitoring of chemical effects on
estrous cyclicity, which can provide critical information on the effects of reproductive
competence. This abstract does not necessarily reflect U.S. EPA policy.
2099 DIETARY IODIDE DEFICIENCY AND DISRUPTION OF
THE HYPOTHALAMIC-PITUITARY-THYROID (HPT)
AXIS BY PERCHLORATE IN ADULT RATS.
B. Fatuyi 1 , K. Jones 1 , E. McLanahan 2 , J. McDougal 3 , B. Blount 4 , L. Valentin-
Blasini 4 , K. Kurunthachalam 5 , T. Kunisue 5 , W. Henderson 6 and J. W. Fisher 1 .
1
College of Public Health, University of Georgia, Athens, GA, 2 National Center for
Environmental Assessment, U.S. EPA, Research Triangle Park, NC, 3 Pharmacology
and Toxicology, Wright State University, Dayton, OH, 4 CCEHIP/NCEH, CDC,
Atlanta, GA, 5 Wadsworth Center, NYS Department of Health, Albany, NY and
6
National Exposure Research Laboratory, U.S. EPA, Athens, GA.
The consequences of perchlorate disruption of the HPT axis by blocking thyroidal
uptake of iodide are considered to be dependent upon iodide in the diet. The goal
of this research was to compare perchlorate-induced disruption of the HPT axis in
the iodide deficient (ID) and sufficient (IS) adult Sprague-Dawley rat. One group
of rats (n=40) was placed on IS chow (193 ng/g) and another (n=40) on ID chow
(16.2 ng/g) for either 54 or 68 days. On Day 53 one-half of the rats from each
group were started on drinking water with a high dose of perchlorate (10
mg/kg/day). The serum iodide levels in the ID rats were 83% lower that the IS rats.
After 54 days of ID diet, serum levels of TT4 were slightly decreased, but thyroid
gland rT3, T3 and T4 levels were decreased 57 to 74% and thyroid weight increased
44%. After 68 days of ID diet, serum TT4 levels dropped 53% while TT3
(+9%) and TSH (+300%) levels increased. Thyroid gland hormones remained decreased
by 67 to 83%. One day of perchlorate exposure did not significantly alter
the existing condition of the HPT axis for the IS or ID groups; however, 14 days of
perchlorate exposure disturbed the HPT axis of both groups. For the ID rats, serum
TT4 was not detectable, TT3 levels were reduced by 80% and TSH levels increased
270%. rT3, T3 and T4 in the thyroid glands were only detected in a few samples at
very low levels. IS perchlorate-treated rat serum TT4 (-60%) and TT3 (-24%) levels
were reduced and serum TSH levels increased 276%. Thyroid hormone levels in
the thyroid gland were reduced 84 to 96%. (Support: U.S. EPA STAR Cooperative
Agreement R832134. This work does not necessarily reflect EPA policy.)
2100 BONE AS A TARGET TISSUE IN THE TOXICOLOGICAL
ASSESSMENT OF ANTI-DIABETES DRUG CLASS OF
SGLT1 INHIBITORS.
T. Kissner 2 , N. Doyle 1 , R. Samadfam 1 , E. Krupp 2 , M. Heinrichs 2 , S. Haile 1
and S. Y. Smith 1 . 1 Toxicology, Charles River Laboratories, Preclinical Services (PCS-
MTL), Senneville, QC, Canada and 2 Sanofi-Aventis Deutschland GmbH, Frankfurt,
Germany. Sponsor: M. Vézina.
Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia
caused by defective insulin secretion, resistance to insulin action, or a combination
of both. Treating hyperglycemia with drugs that block intestinal glucose uptake and
renal glucose reabsorption via the sodium-glucose transporters (SGLT1, SGLT2)
represents a novel approach to diabetes treatment. Calciuria and increased trabecular
bone was found in toxicity studies with the mixed SGLT1/2 inhibitor SAR7226
and the low-absorbable and selective SGLT1 inhibitor SAR474832. To investigate
whether renally excreted calcium was mobilized from bone a specific study in
young vs. old rats was conducted with SAR7226 focusing on electrolytes (serum
and urine), bone quality parameters (pQTC, DXA, bone biomechanical testing),
hormones related to calcium homeostasis, and biomarkers of bone turnover.
Treatment with SAR7226 resulted in dose-related, marked glucosuria –the anticipated
pharmacological effect– polyuria, and calciuria. Normal serum calcium was
maintained while serum phosphorus was slightly increased. PTH and 1,25 vitamin
D3 were markedly suppressed as well as markers of bone turnover. These effects
were associated with increases in bone mineral density (BMD). Increases in BMD
at the spine were positively associated with increases in bone strength. Effects on
bone mass were characterized microscopically by increases in trabecular bone. The
SGLT1/2 inhibitor SAR7226 markedly influences calcium and phosphorus homeostasis
with positive effects on bone mass and strength. Similar effects were observed
with the low-absorbable selective SGLT1 inhibitor SAR474832. From these
results it is concluded that calciuria during studies with SAR7226 and SAR474832
is not associated with a calcium release from bone. These studies highlight the importance
of considering evaluations of the skeleton in the safety assessment of compounds
affecting calcium homeostasis to provide important safety data.
446 SOT 2010 ANNUAL MEETING