The Toxicologist - Society of Toxicology

accepted for regulatory purpose. In vivo, neurotoxicological assessments exploit the
fact that activity of neurons in the central and peripheral nervous system has functional
consequences. The microelectrode array (MEA) assay consists of a culture
chamber into which an integrated array of microelectrodes is capable of measuring
extracellular electrophysiology (spikes and bursts) from electro-active tissues. A
wide variety of electrically excitable biological tissues may be placed onto the chips
including primary cultures of nervous system tissue. Recordings from such an in
vitro cultured system are non invasive, label free evaluations and provide a higher
throughput than conventional electrophysiological techniques. In this fist study 21
blinded substances were tested in a dose-response curve on embryonic rat cortical
neuronal networks on a MEA for their toxicity. The experimental procedure consisted
in the evaluation of the firing activity (spiking rate) and modification/reduction
in response to the chemical administration. Native/reference activity, 30 minutes
of activity recording per dilution, plus the reversibility/recovery points (after
24 hours) were recorded. The IC50 values were calculated using Hill Equation
Fitting tool of the averaged data. The preliminary data, using a set of chemicals
with different mode-of-actions (12 known to be neurotoxic, 2 non neuroactive and
not toxic and 7 not neuroactive but toxic) show a good predictivity (sensitivity:
0.93; specificity: 0.57; predictivity: 0.81). Thus, the MEA with a neuronal network
has the potency to become a powerful tool to evaluate the neurotoxicity of substances
in vitro.
1021 EFFECT OF METALS ON β-ACTIN AND TOTAL
PROTEIN SYNTHESIS IN CULTURED HUMAN
INTESTINAL EPITHELIAL CELLS.
A. R. Calabro and F. A. Barile. Pharmaceutical Sciences, St. John’s University College
of Pharmacy, Queens, NY.
As an important structural protein, β-actin is associated with anchoring of tight
junctions (TJs) to the cell scaffold. Caco-2 cells, an immortal intestinal epithelial
cell line, rely on β-actin to form intact monolayers with high transepithelial electrical
resistance in cell culture inserts. We examined the effect of six metals on expression
of β-actin mRNA and β-actin synthesis, on total and net production of newly
synthesized proteins, and on paracellular permeability (PP) of TJ markers, in confluent
monolayers of Caco-2 cells. [ 3 H]-glycine and [ 3 H]-tyrosine were used as indicators
of newly synthesized proteins in the absence or presence of increasing concentrations
of arsenic, cadmium, copper, manganese, mercury and nickel.
Monolayers were exposed to each metal for 24-hr as well as continuous daily repeated
exposures for 48- and 96-hr. Results suggest that decreases in newly synthesized
proteins, in which β-actin represents about 10%, correlated with 2- to 5- fold
higher expression of β-actin mRNA for the higher concentrations of metals.
Interestingly, IC 50 s calculated for each chemical for 24-hr acute and 48- and 96-hr
repeated dosing experiments, using the MTT viability assay and PP markers, decreased
newly synthesized and total proteins to 10% and 40% of control, respectively.
Overall, the results indicate that, at equivalent concentrations, the metals affect
β-actin mRNA and newly synthesized proteins before cell viability and PP are
compromised. Consequently, the data contributes to elucidating the mechanisms
of metal cytotoxicity that lead to understanding the relationship between TJ integrity,
paracellular transport, and cell viability. (Supported in part by a grant from
the International Foundation for Ethical Research, Chicago, IL; No. 35262-985).
1022 FETAL PHTHALATE SCREEN: ASSESSMENT OF
SEVERAL PHTHALATE ESTERS (PE) ON FETAL
RODENT TESTOSTERONE (T) PRODUCTION AND
GENE EXPRESSION FOLLOWING IN UTERO
EXPOSURE.
C. Lambright 1 , J. Furr 1 , M. Cardon 1 , B. Hannas 2 , D. Bermudez 5 , N. Wrench 3 ,
V. Wilson 1 , P. Foster 4 and E. Gray, Jr 1 . 1 ORD/TAD, U.S. EPA, Durham, NC,
2 NRC Postdoctoral Fellow, Durham, NC, 3 Student Contractor, Durham, NC, 4 NTP,
NIEHS, Durham, NC and 5 NC State Postdoctoral, U.S. EPA, Durham, NC.
. PE are a large family of compounds used in a wide array of consumer, industrial
and medical products. Studies have shown that in utero treatment with PE such as
diethyl hexyl phthalate (DEHP) during the critical period of fetal reproductive development
produced male reproductive malformations by reducing fetal T production
and gene expression. It has been proposed that PE with straight side chains of
C4 to C6 are likely reproductive toxicants but C3 or shorter and C7 or longer are
not. The goal of this study was to test this by using a relatively rapid in vivo screen
to evaluate a suite of PE for their potential reproductive toxicities. We investigated
the effects of 12 PE on fetal testes T production and gene expression by exposing
time pregnant Sprague-Dawley rats via oral gavage (750 mg/kg/day) from gestational
day (GD) 14-18. On GD 18, testes from three fetuses were collected and cultured
for 3 hours. Medium was collected and T levels measured by RIA. The re-
218 SOT 2011 ANNUAL MEETING
maining testes were pooled by litter, mRNA extracted and gene expression for
Insl3, STAR and Cyp11a was measured. Fetal testes T production was significantly
reduced compared to control following treatment with BBP, DBP, DIBP, DPP,
DiHP, DHeP(diheptyl-), DHP (dihexyl-), DCHP (dicyclo-), and DINP. No effect
on fetal T was seen with DEP, BrDEHP, DOTP or DiNCH. Gene expression of
Insl3, STAR and Cyp11a was significantly reduced as compared to control by some
of the above PE that also reduced T production. Some PE reduced reduce both fetal
testes T production and gene expression with the reduction in T production being
consistently the most robust response. Furthermore, some PE with straight chains
less than C4 or longer than C6 disrupted fetal T synthesis. Disclaimer: This abstract
doesn’t reflect USEPA policy. Supported in part by NTP/NIEHS IA#
RW7592285501-1
1023 DETERMINING ESTROGENIC ACTIVITY IN SERUM
FROM OVARIECTOMIZED RATS TREATED WITH
ENVIRONMENTAL COMPOUNDS USING AN IN VITRO
ESTROGEN-MEDIATED TRANSCRIPTIONAL
ACTIVATION ASSAY (T47D-KBLUC).
N. Wrench 1, 3 , L. Gray 1 , D. S. Bermudez 2 , J. Furr 1 and V. S. Wilson 1 .
1 NHEERL, TAD, U.S. EPA, Durham, NC, 2 Postdoctoral Fellow, North Carolina
State University, Raleigh, NC and 3 Student Contractor, U.S. EPA, Durham, NC.
The use of cell-based assays to quantify low levels of estrogen in human serum is an
accepted method. These assays are more sensitive but less specific than radioimmunoassays
(RIA). Thus, we hypothesized that estrogen responsive T47D-KBluc
cells would detect estrogenic activity in serum from ovariectomized (ovx) rats dosed
with estradiol as well as other estrogenic compounds such as bisphenol A-F (BPAF).
Ovx rats were dosed subcutaneously with a negative control (corn oil), positive control
(1 ug/day estradiol-benzoate; EB) or BPAF (600mg/kg/d) for 3 consecutive
days. At necropsy, blood was collected, centrifuged and the serum was stored at -
80°C. Cells were exposed to rat serum diluted 100-fold in RPMI media, supplemented
with 5% dextran-coated-charcoal stripped fetal bovine serum, at
100ul/well in triplicate. Negative control serum did not induce estrogenic activity.
Dilutions of the rat serum demonstrated a dose responsive decline in estrogenic activity
for both EB and BPAF. Calculated estrogen equivalence (EEQ) values for the
EB samples (run in duplicate) averaged: 1.16ng/ml, 0.37ng/ml, 0.22ng/ml. RIA results
for the EB samples of: 1.78ng/ml, 0.53ng/ml, 0.30ng/ml, respectively, confirmed
the accuracy of the cell assay results. Calculated BPAF equivalents in the rat
serum were: 12861ng/ml, 5092ng/ml, 6951ng/ml. These results demonstrate that
the estrogen-mediated T47D-KBluc cell assay can detect circulating estradiol levels
with sensitivity comparable to an RIA, and can be used to quantify estrogenic activity
in serum from rats treated with BPAF. Disclaimer: This abstract does not necessarily
reflect EPA policy.
1024 REPRODUCTIVE ENDPOINTS OF FEMALE WISTAR
RATS EXPOSED TO TESTOSTERONE IN UTERO AND
DURING LACTATION.
M. T. Guerra 1 , R. F. Silva 2 , M. Sanabria 2 , A. C. Gaspar 2 and W. G. Kempinas 2 .
1 Unicamp, Campinas, São Paulo, Brazil and 2 Morphology, Unesp, Botucatu, São
Paulo, Brazil. Sponsor: S. Cohen.
Female reproductive disorders have increased in recent years and exposure to environmental
chemicals may contribute to several gynecologic pathologies, especially
when it occurs during critical periods of development. Despite some concern since
the 1970s, the environmental presence of androgens from anthropogenic origin
was only confirmed within the last few years, and compounds with androgenic activity
were found as contaminants in rivers and in animals intended for human
feeding. In humans, the effects of exposure to androgens on development following
medical conditions, such as polycystic ovary syndrome or congenital adrenal hyperplasia,
are still to be studied. The aim of this work was to evaluate reproductive parameters
of female offspring exposed to testosterone propionate (TP) in utero and
during lactation. Pregnant Wistar rats received either corn oil (GI/n=8) or TP at
concentrations of 0.05 mg/kg (GII/n=7), 0.1 mg/kg (GIII/n=9) or 0.2mg/kg
(GIV/n=10), s.c., from gestational day (GD) 12 to post-natal day (PND) 21. The
following parameters were assessed: body weight, anogenital distance (AGD), nipple
counting, external signs of puberty onset (age of vaginal opening and first estrus),
uterus, ovaries, liver, kidneys and pituitary weights, estrous cycle and sexual
behavior. Results showed no effect upon body weight (except for a decrease at
PND22 and after puberty onset in the GIV group), AGD was only increased in
GIV group at PND1, nipple counting and puberty onset were similar among
groups, the weight of the kidneys was only impaired in GIV group at PND75, the