The Toxicologist - Society of Toxicology

exhibited a hyperactive phenotype, behavior in BPA-exposed embryos injected with
ERRγ MO was indistinguishable from unexposed control morphants. To further
characterize the transcriptional events associated with the hyperactive phenotype,
transcripts associated with ER or ERR signaling were measured by qRT-PCR following
BPA, E2, or GSK4716 exposure. Taken together, we have identified a role
for ERRγ in mediating the neurobehavioral toxicity of developmental BPA exposure.
This research was supported in part by NIEHS T32ES7060, ES00210, and an
EPA STAR Graduate Fellowship to KSS.
2627 EFFECTS OF RADIO FREQUENCY RADIATION
EXPOSURE FROM MOBILE PHONES ON BRAIN
MICROVESSEL ENDOTHELIAL CELLS, A MODEL OF
BLOOD BRAIN BARRIER: AN IN VITRO STUDY.
E. Cuevas 1 , S. M. Lantz 1 , W. J. Trickler 1 , B. R. Robinson 1 , G. D. Newport 1 , P.
C. Howard 2 , N. J. Walker 3 , M. Wyde 3 , A. Desta 4 , M. G. Paule 1 and S. F. Ali 1 .
1 Neurochemistry Laboratory, Division of Neurotoxicology, NCTR/U.S. FDA, Jefferson,
AR, 2 Office of Scientific Coordination, NCTR/U.S. FDA, Jefferson, AR, 3 National
Toxicology Program, NIEHS, Research Triangle Park, NC and 4 ODE, CDRH/U.S.
FDA, Sliver Spring, MD.
The association between exposure to radio frequency (RF) energy similar to that
emitted by mobile phones and potential adverse effects to the central nervous system,
including blood-brain barrier (BBB) dysfunction, neurotoxicity and brain tumors
continue to be an area of scientific controversy. We have designed studies to
test the effects of RF energy exposures on rat and bovine primary brain microvessel
endothelial cells (BMEC). BMEC cells were isolated from fresh rat and bovine cerebral
cortices and cultured in 35 mm petri dishes (12-14 days). The BMEC (80%
confluent) were exposed to RF energy at the specific absorption rate (SAR) levels of
5.04 W/kg (rat) or 5.98 W/kg (bovine) (600sec - 60sec; on-off cycle) for 4, 8, or 24
hours. (The maximum permissible SAR from mobile phones in US is 1.6 W/kg).
Cell viability was evaluated by lactate dehydrogenase (LDH) assay and MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Nitric oxide
(NO) changes were determined by Griess reaction. Heat shock proteins (HSPs), receptor
for advanced glycation end products (RAGE) expression, and inflammatory
makers [tumor necrosis factor-alpha (TNFα), interleukin 1 beta (IL-1β), interleukin
2 (IL-2), and prostaglandin E2 (PGE-2)] levels were evaluated. Under these
conditions, SAR exposures produced no cytotoxic changes as measured by LDH,
MTT and NO, however, the same SAR exposure increased activation of HSP27,
HSP90, and RAGE, and changes in several inflammatory markers (TNFα, IL-1β,
IL-2, and PGE-2) at 8 and 24 hours. These preliminary results indicate that SAR
exposure activates HSPs family, neuroinflammatory markers which may reflect
early intracellular signaling pathways and may ultimately alter BBB integrity. These
studies were supported by NTP/NCTR IAG 224-07-007.
2628 REARING CONDITIONS DIFFERENTIALLY AFFECT
THE LOCOMOTOR BEHAVIOR OF LARVAL
ZEBRAFISH, BUT NOT THEIR RESPONSE TO
VALPROATE-INDUCED DEVELOPMENTAL
NEUROTOXICITY.
D. Zellner 1 , B. Padnos 2 , D. L. Hunter 2 , R. C. MacPhail 3 and S. Padilla 2 .
1 Meredith College, Raleigh, NC, 2 ISTD, U.S. EPA, Res. Tri. Pk., NC and 3 TAD,
U.S. EPA, Res. Tri. Pk., NC.
Zebrafish (Danio rerio) are widely used in developmental research, but little is
known about the role environment may play in their development. Zebrafish are a
highly social organism; thus exposure to or isolation from social environments may
have profound effects. Details of rearing conditions are often sparse in the literature.
This study compared (1) the activity of larval zebrafish that were raised individually
or in groups, and (2) the effect of the developmental neurotoxicant valproate.
We randomly assigned embryos to isolative or inclusive social environments
from 0 to 5 days post fertilization (dpf), while treating them with or without valproate
(50 uM) from 0-2 dpf resulting in a total of four groups (group control,
group treated, single control, single treated). At 5 dpf all embryos were transferred
to singly-housed environments where they remained through locomotor testing (alternating
periods of light and dark) conducted on day 6. Larvae that had been
raised in groups had higher levels of activity than larvae that had been raised individually,
but only in the dark periods. Valproate increased activity in the dark in
both the singly and group housed groups. Further analyses indicated rearing condition
did not significantly affect larval responses to valproate. These results indicate
that rearing conditions affect behavioral development of zebrafish larvae, but that
rearing conditions may not affect the effect of a developmental neurotoxicant. This
is an abstract of a proposed presentation: the information does not necessarily reflect EPA
policy.
2629 ASSESSMENT OF CHEMICAL EFFECTS ON NEURITE
OUTGROWTH, NEURONAL POLARIZATION, AND
SYNAPTOGENESIS IN RAT CORTICAL NEURONS
USING HIGH CONTENT IMAGE ANALYSIS.
B. L. Robinette, J. A. Harrill and W. R. Mundy. Integrated Systems Toxicology
Division, NHEERL, U.S. EPA, Research Triangle Park, NC.
There is a need for efficient, cost-effective methods for screening and prioritization
of potential developmental neurotoxicants. One approach uses in vitro cell culture
models that can recapitulate the critical processes of nervous system development.
In vitro, primary cultures of neocortex undergo a series of morphological changes
including neurite outgrowth, polariation of neurites and synaptogenesis similar to
that observed in vivo. In the present work, primary cultures were prepared from rat
neocortex in a 96-well plate format. A combination of immunocytochemical labeling
and high-content image analysis (HCA) protocols were developed in order to
quantify the time course of neurite outgrowth (βIII-tubulin), neuronal polarization
(βIII-tubulin and MAP2 or neurofilament) and synapse formation (MAP2 and
synapsin). Neurite outgrowth occurred rapidly: 75 % of neurons developed neurites
by 24 h. At this time, ~20% of neurons were axon bearing. An increase in total
neurite length continued up 5 days in vitro (DIV), at which time a change in the
cytoplasmic distribution if MAP2 was observed. The ratio of βIII-tubulin and
MAP2 labeled neurite lengths (NPR) increased from 1.35 to 2.12 between 24 and
120 h, indicative of neuronal polarization. An increase in synapse number was observed
between 6 and 15 DIV. The concentration-response of model chemicals
known to inhibit each process was then examined. K252a (0.01 - 1 μM) applied at
2 h inhibited total neurite outgrowth (20 %), specifically axon outgrowth (97 %),
at 24 h and inhibited neuronal polarization at 120 h (NPR = 1.3). In addition,
mevastatin applied at 9 DIV (0.3 – 30 μM) decreased synapse number at 15 DIV
(~35 %). Effects occurred in the absence of overt cytotoxicity. These data demonstrate
that HCA using a single culture system can be used to screen for chemical effects
on multiple processes of nervous system development. This abstract does not
necessarily reflect USEPA policy.
2630 INITIATING EFFECTS OF METHYLEUGENOL IN
RAT LIVER.
M. J. Iatropoulos, A. M. Jeffrey, J. Duan and G. M. Williams. Department of
Pathology, New York Medical College, Valhalla, NY.
Methyleugenol (ME), a constituent of basil, is hepatocarcinogenic in rodents. To
study initiating effects of ME in rat liver, groups of male F344 rats were administered
by gavage 0 (control, C), 62 (low dose, LD), 125 (mid dose, MD), or 250
(high dose, HD) mg/kg bw/dose, 3 times/week for 8 or 16 weeks (providing cumulative
doses (CD) of 185, 275 or 750 mg/kg bw/week similar to the weekly CD of
the NTP ME bioassay). After week 16, half of the rats were fed 500 ppm phenobarbital
in the diet (+PB), and the rest were maintained on C diet (-PB) for 24
weeks. At 8 weeks, the MD and HD groups had bw reductions, and group relative
liver weight increases. 32 P-nucleotide postlabeling (NPL) revealed 3 adducts in the
livers of all ME groups. The hepatic DNA-adduct values were dose related and were
increased in all ME groups compared to C. PCNA immunohistochemistry (IHC)
revealed that the group hepatocellular replicating fraction (RF) in all ME groups
was increased compared to C. Only the HD rats had hepatocellular altered foci
(HAF), identified by glutathione S-transferase-placental form IHC. At 16 weeks,
there were no differences in bw between groups and no changes in measured serum
liver enzymes. All ME groups had dose-related increases in DNA adducts in both
liver and white blood cells (WBCs) compared to C, although the WBC levels were
1% of those of the liver. The group RF and HAF in all ME groups were increased
compared to C in a dose-related pattern. At the end of the promotion/maintenance
phase (weeks 16-40), the NPL values in both ME/+PB and ME/-PB groups were
lower than the respective 16-wk NPL values, indicating DNA repair. HAF were evident
in all +PB groups, including C. The multiplicity and severity of HAF was
similar between C (1 and 1+, respectively) and LD (2 and 1+) while those of the
MD (8.3 and 3+) and HD (28.8 and 4+) were increased. Hepatocellular adenomas
were evident in a dose-related pattern in both +PB and -PB MD and HD groups,
attaining higher multiplicity and severity in the +PB groups. Thus, ME had initiating
activity and the LD was a NOAEL for pre- and neoplasia, but not for DNA
adducts.
SOT 2011 ANNUAL MEETING 563