The Toxicologist - Society of Toxicology

107 Endocrine Disruption: Weight of the Evidence for Low-Dose
Effects of TCDD on Sperm Counts.
K. L. Hentz, A. L. Williams and J. C. Lamb. Exponent, Alexandria, VA.
Considerable debate is ongoing regarding the potential for endocrine-mediated
low-dose effects. One of the examples that has been presented as evidence for low
dose effects is exposure to 2,3,7,8-tetrachlodibenzodioxin (TCDD) and the impact
on sperm counts in the male offspring. Although one epidemiological study has reported
reduced sperm counts in males exposed to TCDD, the results of this study
are limited by the small number of subjects, evaluation of a single sperm sample,
potential differences in sexual activity, and lack of control in the collection/transport
of the sperm sample. We have conducted a weight-of-the-evidence review of
the toxicological studies investigating the impact of maternal TCDD exposure on
sperm counts in their offspring and the results have been highly variable.
Epididymal sperm counts have been reported to be reduced at doses as low as 64 ng
TCDD/kg, but other studies have failed to show effects at doses of 800 ng
TCDD/kg or higher. Several other animal studies have not reported effects on
sperm counts at doses between 10 and 50 ng TCDD/kg, suggesting a potential
threshold. While sperm effects have been reported in experimental animal studies at
64 ng TCDD/kg, this dose should not be considered a low dose because human exposures
are much lower. This dose is not relevant to current human exposures to
TCDD based on estimated intakes that are 100-fold lower for all dioxins and furans
combined based on TEQ (toxic equivalents). Thus, discussions regarding the
potential endocrine disruptive effects of TCDD on sperm counts are misleading
and do not occur at the low doses experienced by general American public.
108 Effects of Pesticides on Cytochrome P450 17 (CYP17) and
Androgen Receptor (AR) Function in Human H295R
Adrenocortical and LNCaP Prostate Cancer Cells.
C. Robitaille, P. Rivest and T. Sanderson. INRS-Institut Armand-Frappier, Laval,
QC, Canada.
Exposures to endocrine disrupting chemicals, including pesticides, are thought to
be contributing to the increased incidence of certain endocrine-related cancers in
the human population, such as prostate cancer. Prostate cancer growth is initially
androgen-dependent and under control of the nuclear androgen receptor (AR),
which increases the gene transcription of proteins involved in cell proliferation,
such as prostate specific antigen (PSA). CYP17 is a key enzyme in the biosynthesis
of androgens and increased expression is associated with increased prostate cancer
risk. We evaluated the effects of several pesticides suspected or known to modulate
hormonal function in androgen-dependent LNCaP human prostate cancer cells
and in an in vitro steroidogenesis model, the H295R human adrenocortical carcinoma
cell line. Benomyl, vinclozolin and prochloraz reduced dihydrotestosterone-
(DHT)-stimulated LNCaP cell proliferation and nuclear AR protein accumulation
concentration-dependently (0.1-30 μM). Levels of an active phosphorylated form
of AR, pAR-Ser81, were increased by 10 nM DHT, whereas benomyl, vinclozolin
and prochloraz decreased these stimulated levels (after a 1 or 6 h exposure). All
three pesticides reduced DHT-stimulated PSA secretion by LNCaP cells. We found
AR to be expressed in H295R cells but levels of AR and pAR-Ser81 were not affected
by DHT, although they were increased by 30 μM atrazine. Benomyl and vinclozolin
(10 and 30 μM) and prochloraz (1 and 3 μM) decreased CYP17 mRNA
expression in H295R cells at sub-cytotoxic concentrations and prochloraz strongly
inhibited CYP17-catalyzed conversion of pregnenolone to DHEA (24h exposure).
In H295R and LNCaP cells, CYP17 protein expression was increased by atrazine
(30 μM). In conclusion, certain pesticides exert combined antiandrogenic effects in
androgen-dependent prostate cancer cells at the level of CYP17 and AR, the latter
by reducing AR phosphorylation at serine 81.
109 Metabolomics Characterization of the Effects of Estradiol in
Human Breast Cancer Cell Lines.
L. Zhao 1 , S. Odwin-DaCosta 1 , M. M. Vantangoli 2 , H. T. Hogberg 1 ,
M. Bouhifd 1 , A. Kleensang 1 , L. Smirnova 1 , K. Boekelheide 2 , J. D. Yager 1 and
T. Hartung 1 . 1 Department of Environmental Health Sciences, Johns Hopkins
University, Bloomberg School of Public Health, Baltimore, MD; 2 Department of
Pathology and Laboratory Medicine, Brown University, Providence, RI.
Estradiol (E2 or 17β-estradiol) is the most potent naturally occurring estrogen. Its
effects on cell function during development and in adults in a variety of tissues are
mediated through estrogen receptors located in the nucleus, cytoplasm, mitochondria
and at the cell membrane. Our objective is to employ high-throughput liquid
chromatography mass spectrometry based metabolomics analysis to map pathways
affected by estradiol in MCF-7 and T47D cells and then compare them with pathways
affected by endocrine disrupting compounds (e.g. BPA and others).
22 SOT 2013 ANNUAL MEETING
Concurrent cell growth and gene expression studies are also being carried out. Cells
were exposed to estradiol at different concentrations and times. Stimulation of cell
growth was observed at 0.1nM in association with changes in the metabolome at 6
and 24hrs. A time course study at 100nM estradiol revealed that some metabolites
were significantly altered at various time points. The metabolites and pathways
most affected included metabolites from cellular energy-related pathways (e.g.
Malate, Fumarate, Pyruvate) and amino acid synthesis pathways (e.g. Arginine,
Valine, Ornithine, Leucine/Isoleucine, 2-Methylmalate). Further investigation is
underway and should give more insight into the identification of metabolism pathways
affected by estradiol and endocrine disruptors.
110 Creating In Vitro Test Methods for Endocrine Disruptor
Risk Assessment: Assay Development.
C. Le Sommer, S. M. Ross, P. D. McMullen, M. E. Andersen and R. A. Clewell.
The Hamner Institutes, Research Triangle Park, NC.
Endocrine disruptor test programs in the US are moving forward to identify active
compounds and prioritize them for subsequent in-life toxicity studies. Our focus is
on developing in vitro tests for cellular responses to endocrine active substances that
will be sufficient for health risk assessment without moving on to in-life toxicity
tests. We have begun a research effort for the estrogen receptor (ER) pathway to: 1)
map signaling pathways for estrogen mediated proliferation, 2) define the dose-response
for perturbation of estrogen signaling by xenobiotics and 3) develop computational
models to predict chemical effect on uterine epithelium. The first phase
develops a cell-based assay in human Ishikawa endometrial cell line expressing the
three major ERs: ESR1, ESR2 and G-protein coupled receptor GPER. The doseresponse
for 17β-estradiol (E2) and 17α-ethynyl estradiol (EE) (0.001 – 10 nM)
were examined at 1, 2, 3, 4, 5, and 6 days of exposure for proliferation and induction
of protein and gene targets of ESR1, including alkaline phosphatase (ALP),
proliferation associated gene GREB1 and progesterone receptor (PGR). E2 and EE
increased proliferation and ALP activity by day 3 at 0.01 nM, while gene induction
(GREB1, ALP, PGR) occurred earlier (day 1). Cells were also treated with selective
receptor agonists PPT, DPN, and G1 targeting ESR1, ESR2 and GPER. PPT increased
proliferation, ALP activity and gene expression similar to EE. DPN induced
proliferation and ALP only at higher doses associated with cross-reactivity
with ESR1. GPER plays a role in regulating uterine proliferative response; however,
we saw no change in proliferation upon treatment with G1 alone. This may result
from low GPER expression in our cells. We also conducted expression analysis of
studies with Ishikawa cells from our laboratory and published sources to evaluate
dose-response of GO-categories by these receptor specific ligands. In vitro responses
have been analyzed in light of in vivo rodent uterotrophic assay transcriptomic data
to show consistency between in vitro and in vivo assays.
111 Leaching of Chemicals with Estrogenic Activity from
Packaging into Popular Lab Animal Feeds.
D. Klein 1 , C. Z. Yang 2 , G. J. Kollessery 2 and G. D. Bittner 1, 2, 3 . 1 PlastiPure, Inc.,
Austin, TX; 2 CertiChem, Austin, TX; 3 University of Texas, Austin, TX. Sponsor: M.
Stoner.
A robotized MCF-7 cell proliferation assay currently undergoing validation by IC-
CVAM/NICEATM was used to quantify the total estrogenic activity (EA) of chemical
mixtures leaching from lab animal feed bags into feed over time. A standard
open-formula (low phytoestrogen AIN-93G) feed was purchased from Harlan®,
Research Diets, and TestDiet®. The TestDiet® feed was packaged in EA-Free
bags recently developed by PlastiPure for PMI® LabDiet® and other customers.
All diets were analyzed 0, 2, and 4 weeks after purchase. All leachates were quantified
relative to the percentage of the maximum DNA produced per well induced by
a test chemical with respect to the maximum DNA produced per well induced by
the 17beta-estradiol positive control (%RME2) and corrected by the cell response
to the vehicle (negative) control. The significance level (p < 0.01) to detect EA was
a %RME2 value 3 standard deviations more than the VC. According to this conservative
criterion, EA levels in 72 hour ethanol extracts of commercial bags for
TestDiet®, Harlan®, and Research Diets were non-detectable (ND), significantly
positive, and significantly positive, respectively. In the feed study, there was
no detectable EA activity in TestDiet® feed at any time point. Feed samples from
Harlan® and Research Diets were ND at week 0 but significantly positive following
storage for 2 and 4 weeks after purchase. EA readings for Harlan® and
Research Diets increased from week 0 to week 2 and again from week 2 to week
4. These data suggest that leaching of chemicals having EA from the non-EA free
plastic packaging can alter the hormonal activity of semi-purified animal feeds.
Such leaching of chemicals having EA from plastic packaging into animal feeds may
impact research protocols that examine hormonally- responsive end-points.