The Toxicologist - Society of Toxicology

morigenesis. Knocking down of JARID1A increased H3K4 tri-methylation at the
promoters of heme oxygenase-1 (HMOX1) and decay accelerating factor (DAF)
genes. These results indicate that hypoxia may target JARID1A which in turn increases
H3K4 tri-methylation at both the global and gene specific levels, leading to
the altered programs of gene expression and tumor progression.
154 ROLE OF EPIGENETIC MECHANISMS IN
DIFFERENTIAL REGULATION OF THE DIOXIN-
INDUCED HUMAN CYP1A1 AND CYP1B1 GENES.
S. R. Beedanagari and O. Hankinson. Molecular Toxicology, UCLA, Los Angeles, CA.
The Aryl Hydrocarbon Receptor (AHR)/ARNT heterodimer mediates carcinogenesis
by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) and certain polycyclic
aromatic hydrocarbons (PAHs) by activating several genes, including
CYP1A1 and CYP1B1. Metabolism of PAHs by CYP1A1 and CYP1B1 plays a
major role in carcinogenesis mediated by these compounds. Epigenetic mechanisms
involved in the dioxin–induced differential regulation of the human CYP1A1 and
CYP1B1 genes in the MCF-7 and HepG2 cancer cell lines were investigated. RNA
PolII, but not AHR recruitment correlated with the dioxin-induced CYP1A1 and
CYP1B1 mRNA expression. Using the DNA methyltransferase inhibitor, 5-
AzadC, and bisulfite sequencing, we demonstrated that CYP1B1 mRNA expression
is silenced in HepG2 cells due to promoter hypermethylation. Four chromatin
modifications of nine studied, AcH3K9, AcH3K14, AcH4, and me3H3K4
strongly correlated with dioxin-induction of the CYP1A1 and CYP1B1 genes in
MCF-7 and HepG2 cells. We also demonstrated that hypermethylation of the
CYP1B1 promoter can not inhibit coactivator recruitment. Finally the knock down
of coactivator protein, p300 significantly inhibited dioxin-inducibility of these
genes in MCF-7 cells, and diminished the extent of the above four chromatin modifications
of these genes. In conclusion, these data demonstrate a role for chromatin
modification in transcriptional regulation of the CYP1A1 and CYP1B1 genes and
a role for DNA methylation in silencing the CYP1B1 gene in HepG2 cells.
155 THE EFFECTS OF DEVELOPMENTAL EXPOSURE TO
PCBS AND/OR PBDES ON AUDITORY FUNCTION.
E. Poon 1 , B. E. Powers 2 , R. M. McAlonan 1 and S. L. Schantz 1 . 1 Univerisity of
Illinois, Urbana, IL and 2 Cornell University, Ithaca, NY.
: Human and animal studies have provided evidence that developmental exposure
to polychlorinated biphenyls (PCBs) causes hearing loss. Polybrominated diphenyl
ethers (PBDEs) are structurally similar to PCBs and have similar neurotoxic effects,
including reducing thyroid hormone levels. There is strong evidence that reductions
in thyroid hormone levels lead to auditory deficits. This study attempted to
see if developmental exposure to PBDEs caused hearing loss, and if a combination
of PBDEs and PCBs produced an additive effect on hearing loss, possibly via a reduction
of circulating thyroid hormone during the critical stage for cochlear development.
We examined auditory function in adult rats exposed to PCBs and/or
PBDEs during gestation and lactation. Dams were orally exposed to 3 or 6
mg/kg/day of PCBs, 5.7 or 11.4 mg/kg/day of PBDEs (molar equivalents of the
PCB doses), or combinations of 3 mg/kg/day PCBs + 5.7 mg/kg/day PBDEs or 6
mg/kg/day PCBs + 11.4 mg/kg/day PBDEs. Auditory function was assessed when
the pups reached adulthood by measuring distortion product otoacoustic emissions
(DPOAEs), which evaluate the integrity of the outer hair cells of the cochlea.
Serum thyroid levels were measured at weaning. DPOAE thresholds were elevated
and DPOAE amplitudes were decreased across all frequencies in the 6 mg/kg PCB
group, demonstrating a significant hearing impairment. PBDEs alone did not result
in any significant hearing loss. Both the low and high combined exposures resulted
in lower DPOAE amplitudes and higher thresholds across all frequencies. In
summary, we found no evidence of hearing loss following early PBDE exposure.
However, the PBDE doses used in this study did not reduce circulating thyroid
hormone levels as dramatically as the molar equivalent PCB doses and this could
explain the absence of hearing impairments following exposure to PBDEs alone.
Research was funded by NIEHS grants R01 ES015687, T32 ES007326, and the
Merck-Merial Veterinary Scholars Program.
156 RODENT AND HUMAN NEUROPROGENITOR CELLS
FOR HIGH-CONTENT SCREENS OF CHEMICAL
EFFECTS ON PROLIFERATION AND APOPTOSIS.
M. E. Culbreth 2 , T. M. Freudenrich 1 , B. Robinette 1 , J. A. Harrill 1 , W. R.
Mundy 1 and T. J. Shafer 1 . 1 ISTD, U.S. EPA, Research Triangle Park, NC and
2
Biochemistry, NC State University Raleigh, NC.
The objective of these experiments is to develop high-throughput screens for proliferation
and apoptosis in order to compare rodent and human neuroprogenitor cell
responses to potential developmental neurotoxicants. Effects of 4 chemicals on proliferation
and apoptosis in mouse cortical neural stem cells (mCNS) and ReNcell
CX Cells (ReN), an immortalized human cortical neuroprogenitor cell line, were
examined using automated high content image analysis. Expression of neuroprogenitor
cell markers Nestin and Sox2 were confirmed in proliferating cultures of
both cell types. Cells were plated in 96-well plates and exposed (24 hrs) to 4 chemicals
with known actions on proliferation and apoptosis; aphidicolin, staurosporine,
etoposide, and actinomycin D. Both endpoints were immunohistochemically assessed
with antibodies to BrdU in replicating DNA, activated p53 and activated
Caspase 3. In both cell types, aphidicolin (1, 3, 10 μM) decreased BrdU intensity
to 40-60% control and increased p53 and Caspase 3 intensity to < 200% control.
Staurosporine (0.03, 0.1, 0.3 μM) decreased BrdU intensity to 35% control for
mCNS and ReN, but increased p53 intensity 200-400% control in both cell types.
Caspase 3 intensity decreased to 50% control in mCNS, but not ReN cells.
Staurosporine also decreased the number of cells/well. In both cell types, etoposide
(0.001, 0.01, 0.1 μM) decreased BrdU and Caspase 3 to ~75% and ~80% control,
respectively but not p53 intensity. Actinomycin D, (0.001, 0.01, 0.1 μM) decreased
BrdU intensity to 30% control in mCNS but not ReN cells. By contrast,
p53 and Caspase 3 increased to 400-800% and 400-1000% control, respectively in
both cell types. These results demonstrate aphidicolin and actinomycin are positive
controls for proliferation and apoptosis, and that this high-content assay for proliferation
and apoptosis can be used in both human and mouse neuroprogenitor cells.
(Supported by the SOT-Colgate-Palmolive Alternative Grant Award. This abstract
does not reflect Agency Policy).
157 MARBLE-BURYING BEHAVIOR IN PCB-EXPOSED
MICE DIFFERING AT THE CYP1A2 LOCUS.
B. Hays, R. Floyd, E. Altenhofen, C. Kamau-cheggeh, S. Kraemer and C.
Curran. Biological Sciences, Northern Kentucky University, Highland Heights, KY.
Polychlorinated biphenyls (PCBs) are ubiquitous industrial chemicals banned in
the 1970s and 80s and associated with learning, memory and behavioral changes in
children exposed during development. As concentrations of PCBs decrease in the
environment, we are searching for genetic differences in susceptibility to developmental
PCB exposure with the long-term goal of identifying the most vulnerable
human populations. CYP1A2 has long been known to sequester toxicants such as
dioxin and planar PCBs. Our previous work used Cyp1a2(-/-) mice to examine
PCB-induced learning and memory deficits. We found increased thigmotactic behavior
in the Morris Water Maze and higher baseline locomotor activity. To further
explore differences in behavior, we compared marble-burying behavior in PCBtreated
Cyp1a2(-/-) and wild-type mice, a common measure of anxiety. We found
that PCB-treated Cyp1a2(-/-) mice buried significantly fewer marbles (7.9 ± 1) than
PCB-treated Cyp1a2(+/+) mice (11.3 ± 1) with p