The Toxicologist - Society of Toxicology

effect doses obtained with the rat uterotrophic assay. As a result, a 2-fold more accurate
confidence interval for the curve describing the EE2 equivalent dose - response
curve was obtained. The present methodology increases the in vivo predictive
value of the ER-Calux bioassay for the selected compounds, and could provide
a method of deriving an in vitro effect-based benchmark dose needed for risk assessment
of samples containing estrogenic compounds.
2349 PCB-153 AND BDE-47 INCREASE THYROXINE (T 4 )
CATABOLISM IN RAT AND HUMAN HEPATOCYTES.
V. M. Richardson 1, 2 and M. J. DeVito 3 . 1 ORD/NHEERL/ISTD, U.S. EPA,
Research Triangle Park, NC, 2 Curriculum in Toxicology, UNC at Chapel Hill,
Chapel Hill, NC and 3 Toxicology Branch, NIEHS/NTP, Research Triangle Park, NC .
Previous studies demonstrate that in vivo exposure to 2,2’,4,4’,5,5’-hexachlorobiphenyl
(PCB-153) and 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47) decrease
serum thyroxine (T 4 ) levels in rats. This decrease is thought to occur through
the induction of hepatic metabolizing enzymes resulting in the enhanced catabolism
of T4 . There is some evidence the T4 decrease occurs in humans, but the mechanism
is unclear. Using primary rat and human hepatocytes, we compared the effects
of PCB-153 and BDE-47 on T4 catabolism. 48 hours after plating, fresh
sandwich-cultured human or Sprague-Dawley rat hepatocytes were treated with
PCB-153 or BDE-47 at a concentration of 30uM in 0.1% DMSO. 72 hours after
dosing, cell culture media was replaced with media containing [I125 ]-T4 at median
serum concentrations observed in rats and humans; 0.05uM and 0.1uM, respectively.
24 hours after [I125 ]-T4 administration, media was collected, T4 and its
metabolites were separated by UPLC, and fractions collected and quantified on a
gamma counter. The predominant metabolite found in media of control and
treated hepatocytes was T4-glucuronide (T4G). Results show a higher basal activity
of T4G formation in unexposed rat hepatocytes as compared to unexposed human
hepatocytes (5.2 vs.0.1 fmol T4G/min/mg protein). Following PCB-153 treatment,
T4G formation in rat hepatocytes increased 3.8-fold while increasing 10.8fold
in human hepatocytes. BDE-47 increased T4G formation in rat and human
hepatocytes by 1.6- and 13.3-fold, respectively. The data demonstrates that basal
activity for T4G formation in rat hepatocytes is approximately 50-times higher
than human hepatocytes. Additionally, PCB-153 and BDE-47 increase T4G formation
in rat and human hepatocytes, with greater increases in humans. As a possible
in vitro screening tool for environmental toxicants, the results highlight the
utility of primary hepatocytes in evaluating potential species differences and
human health risk.(This abstract of a proposed presentation, does not reflect
USEPA or NIH policy.)
2350 TRICLOSAN DECREASES RAT THYROXINE: MODE-
OF-ACTION, DEVELOPMENTAL SUSCEPTIBILITY
AND HUMAN RELEVANCE.
K. B. Paul 1, 2 , J. M. Hedge 2 , S. O. Simmons 2 , M. J. DeVito 3, 1 and K. M.
Crofton 2, 1 . 1 Toxicology, University of North Carolina, Chapel Hill, NC, 2 Integrated
Systems Toxicology Division, NHEERL, ORD, U.S. EPA, Research Triangle Park, NC
and 3 NTP, NIEHS, Research Triangle Park, NC .
Triclosan (TCS) decreases rat serum thyroxine (T4). In vivo and in vitro approaches
were used to address three uncertainties: by what mode-of-action (MOA) does TCS
decrease T4; does TCS decrease T4 developmentally; and, are effects observed in
rats relevant to humans? To test the hypothesized MOA that TCS decreases T4 via
activation of the pregnane X and constitutive androstane receptors (PXR, CAR),
and subsequent up-regulation of hepatic catabolism of T4, weanling female Long-
Evans rats received TCS po (0-1000 mg/kg/day) for 4 days. Pentoxyresorufin-Odeethylase
(PROD) and uridine diphosphate glucuronyltransferase (UGT) enzyme
activities were measured in liver microsomes. qRT-PCR was used to measure
mRNA expression of CYPs, UGTs, and sulfotransferases. PROD and UGT activities
increased 8- and 2-fold at 1000 mg/kg/day. Cyp2b2, Cyp3a1, Ugt1a1 and
Sult1c1 mRNA expression levels were induced 2- to 4-fold at 300 mg/kg/day. To
test this MOA in dams and offspring, pregnant Long-Evans rats received TCS po
(0-300 mg/kg/day) from gestational day (GD) 6 to postnatal day (PND) 21. Serum
T4 decreased 30% in GD20 dams and fetuses, PND4 pups and PND22 dams at
300 mg/kg/day. Hepatic PROD activity increased 3-fold in PND22 dams and
PND4 pups, and UGT activity was 1.5-fold in PND22 dams at 300 mg/kg/day.
Reductions of 30% in T4 for dams and GD20 and PND4 offspring with concomitant
increases in PROD and UGT activity suggest TCS may reduce T4 during development
by a similar MOA to weanlings. To assess human relevance of the MOA,
cell lines (Puracyp, Inc.) containing a CYP3A promoter-luciferase gene fusion were
used to test activation of PXR in luciferase-based reporter assays. TCS moderately
activated human PXR, but did not activate rat PXR. The data suggest that the initial
key event of the MOA may be different between rats and humans, but the
downstream effects may be similar between species. This abstract does not necessarily
reflect the policy of the US EPA or NIH.
2351 EVALUATION OF SUBSTANCES ROUTINELY TAKEN
IN EVERYDAY LIFE IN THE H295R
STEROIDOGENESIS ASSAY.
H. Tinwell, S. Colombel and R. Bars. Research Toxicology, Bayer SAS, Sophia
Antipolis CEDEX, France.
In response to the demand for increased regulation of endocrine disrupters (EDs),
assays that have been developed specifically for their identification (both in vitro
and in vivo) are becoming part of the safety evaluation of chemicals. These assays
have undergone extensive validation using known EDs and currently much focus is
being placed on the activity of synthetic chemicals, particularly agrochemicals, in
these tests. However, very little, if any, information is available concerning the activity
of compounds in these ED assays that we knowingly and often willingly expose
ourselves to on a daily basis (eg caffeine, vitamins, painkillers). To address this
absence of data, various substances routinely taken, often on a daily basis, have been
tested in vitro using the H295R steroidogenesis assay. This assay is part of the
OECD level 2 battery of in vitro assays for endocrine disruption and is mandatory
in the current US EPA Endocrine Disruptor Screening Program. The data generated
in this in vitro assay is considered to provide an alert for potential in vivo endocrine
effects via disturbance of sex steroid hormone synthesis and can ultimately
contribute to the subsequent categorization of a compound as an ED. We tested
each compound at concentrations which were not cytotoxic and which were relevant
in terms of possible daily adult human exposure. Our data indicate that whilst
some compounds, such as folic acid and sucrose gave no clear effects on progesterone,
testosterone or estradiol synthesis following 48h incubation with the
H295R cells, other compounds such as caffeine and vitamin C consistently induced
clear, dose related increases in estradiol secretion under the same conditions
of test. In contrast, vitamins B6 and B3 appeared to reduce estradiol secretion.
Evaluation of additional compounds such as paracetamol, ibuprofen and aloe vera
is on-going; however this first dataset indicates the importance of determining and
understanding assay limitations before embarking on data interpretation.
2352 BISPHENOL A DISRUPTS STEROIDOGENIC
PATHWAYS AND ADRENAL AND SEX HORMONE
SECRETION IN THE HUMAN ADRENOCORTICAL
H295R CELL LINE.
A. Oskarsson 1 , Ohlsson 1 , C. Jansson 2 and E. Ullerås 1 . 1 Department of
Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural
Sciences, Uppsala, Sweden and 2 Department of Aquatic Sciences and Assessment,
Swedish University of Agricultural Sciences, Uppsala, Sweden.
Bisphenol A (BPA), a monomer used in production of plastics, has endocrine disrupting
properties through a broad spectrum of mechanisms. It acts on the estrogen,
androgen and thyroid hormone systems by interacting with hormone receptors.
Furthermore, BPA has been shown to affect the synthesis of sex hormones
while less attention has been paid to adrenocortical hormones. We have investigated
the effects of BPA on steroid hormone synthesis in the human adrenocortical
cell line H295R, which expresses all enzymes required for secretion of adrenocortical
and sex hormones. Cells were treated with 0.01-100 μM BPA for 24 hours and
levels of 12 secreted hormones and steroid intermediates were determined by
ELISA and LC-MS/MS. A dose-dependent inhibition of 11-deoxycortisol, cortisol,
DHEA, androstenedione and testosterone secretion was demonstrated with significant
effects starting at 0.1 μM BPA. In contrast, the secretion of progesterone and
estradiol was increased by BPA. Secretion of 11-deoxycorticosterone, corticosterone
and aldosterone was reduced but only at the highest BPA concentration and
17α-OH-progesterone was not affected at all. The observed pattern of effects
might be mediated by an inhibition of the steroidogenic enzymes CYP17A1 and
CYP21A2 together with a stimulation of CYP19A1. Gene expression, analysed by
quantitative RT-PCR revealed a significant down-regulation of CYP17A1,
CYP21A2 and 3βHSD2, but only at the highest concentration of BPA. An up-regulation
in expression of the CYP19A1 gene was observed, although only at 1 and
10 μM BPA. We conclude that BPA disrupts adrenal steroidogenesis, with cortisol
secretion as a sensitive target, as well as synthesis of sex hormones in the human
H295R cell line. The effects can only partly be explained by effects on expression of
steroidogenic genes.
SOT 2011 ANNUAL MEETING 505