The Toxicologist - Society of Toxicology

2644 COVALENT BINDING OF 14 C 2-METHOXY-4-
NITROANILINE IN MALE SPRAGUE-DAWLEY RATS.
R. Snyder 1 , S. Waidyanatha 2 , I. Surh 2 , T. Banks 1 , P. Patel 1 , S. Black 1 and T.
Fennell 1 . 1 RTI International, Research Triangle Park, NC and 2 National Toxicology
Program, National Institute of Environmental Health Sciences, Research Triangle
Park, NC.
2-Methoxy-4-nitroaniline (MNA) is used in dyeing textiles, as a dye in the printing
industry, and as an intermediate in the synthesis of azo dyes that have applications
in tattoo inks, emulsion paints, and toy enamels. MNA has structural similarity to
the known carcinogens, 2-methoxy-5-nitroaniline (5-nitro-o-anisidine), o-anisidine
and 2,4-diaminoanisole. The objective of this study was to evaluate the ability
of MNA-derived metabolites to bind covalently to tissue macromolecules in
rats. [Ring- 14 C] MNA was administered at 150 mg/kg b. wt. to male Sprague
Dawley rats (approximately 220 μCi/rat) by gavage. After 24 h, animals were euthanized
and blood and selected tissues were collected and analyzed for total radioactivity
in tissues, radioactivity bound to tissues and to DNA. In tissues, the total radioactivity
was highest in liver (0.31 ± 0.04 % of the dose) and kidney (0.053 ±
0.021 %). However, the concentration of radioactivity was highest in kidney and
cecum (91 ± 4 and 22 ± 1 μg equiv/g tissue, respectively). Concentrations were
similar in ileum, jejunum, and large intestine (approximately 16 μg equiv/g).
Radioactivity bound determined by exhaustive extraction was highest in liver (43%
of the total in tissue) followed by kidney (13 % of the total in tissue), cecum (12 %)
stomach (9 %), and large intestine (9 %). Binding was low in ileum, jejunum, and
duodenum (1-2% bound). In red blood cells, 27% of the activity was bound compared
with 3% in plasma. Radioactivity associated with DNA (pmol equiv/mg
DNA) was highest in the cecum (160 ± 27 pmol/mg) followed by large intestine
(13 ± 2 pmol/mg), and was not significantly above background in other tissues.
These studies suggest that MNA is metabolized to reactive species that are capable
of binding to macromolecules including DNA. The nature of the metabolites of
MNA is under investigation in companion studies.
2645 DIFFERENTIAL MODULATION OF CANCER-RELATED
MOLECULAR NETWORKS IN HUMAN AND RAT
URINARY BLADDER CELLS EXPOSED TO TRIVALENT
ARSENICALS.
K. Bailey 1 , K. Wallace 2 , S. Thai 2 , D. C. Wolf 2 , S. W. Edwards 2 and R. C. Fry 1 .
1 Environmental Sciences and Engineering, University of North Carolina at Chapel
Hill, Chapel Hill, NC and 2 U.S. EPA, Research Triangle Park, NC.
Arsenic (As) is classified as a known human carcinogen with primary targets of urinary
bladder (UB), skin and lung. The most prevalent source of As exposure in humans
is drinking water contaminated with inorganic As (iAs). The mode of action
(MOA) of As carcinogenesis in target cells is largely undefined, including which arsenical(s)
elicit a carcinogenic response. Two urinary metabolites of iAs,
monomethylarsonous acid and dimethylarsinous acid (MMAIII and DMAIII, respectively),
are attractive candidates as UB carcinogens. We used a transcriptomics
approach to examine the altered molecular pathways and networks in human and
rat UB cells after exposure to individual arsenicals to investigate the MOA of Asdriven
UB carcinogenesis. UROtsa (human) and MYP3 (rat) cells were exposed to
relatively non-cytotoxic (>75% cell viability), environmentally-relevant concentrations
(1 μM) of iAsIII, MMAIII, and DMAIII for 24 h. Differentially expressed
genes were determined for each treatment group relative to controls and analyzed
for statistically significant biological functions, molecular networks and canonical
pathways. Each treatment group elicited a distinct transcriptional profile with few
shared genes in top networks or canonical pathways, although lipid metabolism was
a function associated with all top networks. Distinct from the other groups,
MMAIII exposure in UROtsa cells generated changes in several top molecular networks
that are consistent with pathways suspected to play key roles in human UB
carcinogenesis, suggesting that common factors may drive UB carcinogenesis in humans
and that they may be distinct from those in rats. [This abstract does not necessarily
reflect EPA policy.]
2646 THE EFFECTS OF ORAL TREATMENT WITH
TRANSFLUTHRIN ON THE BLADDER EPITHELIUM
OF RATS AND MICE AND ITS METABOLITE,
TETRAFLUOROBENZOIC ACID ON UROTHELIAL
CELLS IN VITRO.
S. M. Cohen 1 , L. Arnold 1 , S. Lautraite 2 , L. Sheets 2 , S. Wason 2 , B. Stahl 2 , D.
Eigenberg 3 , K. L. Pennington 1 , S. Kakiuchi-Kiyota 1 and M. Yokohira 1 .
1 University of Nebraska Medical Center, Omaha, NE, 2 Bayer AG, Bayer
CropScience, Monheim, Germany and 3 Xenometrics, Stilwell, KS.
Transfluthrin, a pyrethroid insecticide, induced urinary bladder tumors in rats but
not in mice in 2-year bioassays. The mechanism was investigated via an in vivo
study with transfluthrin in rats and mice and in vitro studies to examine the effects
of its major metabolite tetrafluorobenzoic acid (TFBA) on rat (MYP3) and human
(1T1) urothelial cell lines. For the in vivo study, rats were fed diet containing 0,
2000 or 5000 ppm transfluthrin for 4 weeks, 0 or 2000 ppm for 13 weeks, or coadminstered
1.25% NH4Cl with 0 or 5000 ppm transfluthrin to evaluate the effects
of acidification of the urine. Mice were treated with 0 or 1000 ppm transfluthrin
for 4 weeks. After 4 weeks, there was no evidence of hyperplasia or
increased bromodeoxyuridine labeling index in any treatment group. After 13
weeks treatment with 2000 ppm transfluthrin, cytotoxicity and necrosis of the
urothelial superficial layer were detected in the rat bladder by scanning electron microscopy.
The urinary concentration of TFBA in rats fed 2000 ppm transfluthrin
was 2.94±0.67 mM. The LC50 of TFBA was 2.25 mM for MYP3 cells and 2.43
mM for 1T1 cells. These studies support cytotoxicity and regenerative proliferation
as the mechanism for induction of bladder tumors with high oral doses of transfluthrin
due to metabolism of transfluthrin to the weakly cytotoxic TFBA that is
excreted at high concentrations in the urine of rats administered high doses of
transfluthrin (≥2000 ppm) for an extended period.
2647 NEUROBEHAVIORAL ALTERATIONS IN CARG-BOX
BINDING FACTOR-A KNOCK-OUT MICE.
J. T. Barrett 1, 2 , J. R. Richardson 2 , X. Zhang 3 , M. Fang 2 , K. R. Reuhl 2, 4 and H.
Zarbl 2 . 1 GSBS, UMDNJ, Piscataway, NJ, 2 EOHSI, RWJMS, UMDNJ, Piscataway,
NJ, 3 University of Washington, Seattle, WA and 4 Rutgers, Piscataway, NJ.
CArG-box binding factor A (CBF-A) is a member of the heterogeneous nuclear ribonucleoproteins
(hnRNPs) family of RNA-binding proteins that are involved in a
variety of cellular functions. We previously showed that deregulation of CBF-
A/hnRNP A/B is involved in transcriptional regulation of the HA-ras promoter and
contributes to mammary carcinogenesis. Recently, we have developed CBF-A
knock-out mouse models to further study the functions of this hnRNP protein. In
addition to its role in mammary carcinogenesis, CBF-A is also expressed in the
brain and is involved in regulation of immediate early gene expression.
Furthermore, a single nucleotide polymorphism (SNP) in the arginine vasopressin
gene promoter that reduced CBF-A binding was associated with alterations in anxiety
behavior. To directly determine the role of CBF-A in neurobehavior, we assessed
open-field locomotor function and anxiety behavior in an elevated plus maze
for wild type, heterozygous, and CBF-A null mice. For locomotor activity, mice
were allowed to habituate to the open-field for 30 min and ambulatory distance
and vertical counts were calculated over the next 30 min. For the elevated plus
maze, mice were allowed to freely explore the maze for 5 min, and percent time
spent in the open arms was determined. In both tasks, there were no significant differences
between wild-type and mice heterozygous for CBF-A. However, ambulatory
distance traveled was increased by 89% in CBF-A null mice compared to wildtype.
CBF-A null mice also demonstrated a 230% increase in vertical activity. In
the elevated plus maze, CBF-A null mice spent 319% more time in the open arms
of the maze compared to wild-type, suggesting that loss of CBF-A results in a lowanxiety
phenotype. Taken in concert, these data suggest that CBF-A plays an important
role in neurobehavioral function. Supported by R01ES015991 and
P30ES005022
2648 INFLUENCE OF BENZO(A)PYRENE (BAP)
BIOTRANSFORMATION ON BAP-INDUCED
CYTOTOXICITY IN HT-29 HUMAN COLON CANCER
CELLS.
J. N. Myers and A. Ramesh. Biochemistry & Cancer Biology, Meharry Medical
College, Nashville, TN.
The majority of sporadic cancer deaths are attributed to exposure and intake of environmental
toxicants. Benzo(a)pyrene (BaP) is one such ubiquitous environmental
toxicant. Being a combustion byproduct, high levels of BaP have been detected in
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