The Toxicologist - Society of Toxicology

one marrow. Since Chk1 is an attractive Oncology target, Chk1 inhibitors currently
under development should be examined closely for potential bone marrow
toxicity.
1154 RADIATION AND SENESCENCE: DECELERATION OF
CELL-CYCLE IN PRIMITIVE HEMOPOIETIC
PROGENITORS (CFU-S13) WAS ONLY SIGNIFICANT
PARAMETER DURING AGING, WHICH WAS
REACTIVELY ACCELERATED AFTER 2GY WHOLE-
BODY IRRADIATION.
Y. Hirabayashi1 , I. Tsuboi2 , K. Sekita1 , J. Kanno1 , Y. Kusunoki3 and T. Inoue1, 2,
3 . 1NCBCR, NIHS, Tokyo, Japan, 2School of Medicine, Nihon University, Tokyo,
Japan and 3RERF, Hiroshima, Japan.
Toxicology is a function of xenobiotic responses, and aging is a function of xenobiotic
responses during lifetime; thus, “time” during life is an essential toxicologic parameter
for living creatures. Effect of ionizing radiation during lifetime is thus formulated
by a function of simultaneous equations over the effects of “lifetime” and
“radiation”. Owing to the mission of NIAID, the study was conducted to evaluate
a possible additive/ synergic effect of radiation exposure during aging in cell cycling
parameters of the hematopoietic stem (HSC) /progenitor cell (PGC) compartment
(Contract No. HHSN 272 200 900 059C). Kinetics of cell cycles in HSC/PGC
(GM, mature PGC = CFU-GM; S9, less mature PGC = CFU-S9; and S13, immature
PGC = CFU-S13) were evaluated by the BUUV assay (The HSC/PGC-specific
cell kinetics, Hirabayashi Y. et al. Exp Biol Med 227:474-9, 2002; Sf/T, S
phase fract./unit time; DT, Doubl. time; Cf, cycling fract.). During the steady state,
old mice (Om; 23 mos.) showed, as compared with young mice (Ym; 3 mos.), an
increased to a decreased rate of Sf/T in GM ~ S13; a decelerated to an accelerated
DT from GM ~ S13; and an increased to a decreased Cfs from GM ~ S13, respectively.
When both Ym and Om were irradiated with 2Gy and given a recovery time
for 4 weeks, each parameter of both groups showed; the rates of Sf/T were increased
in all PGC from GM ~ S13 in Ym, whereas in Om, rates of Sf/T were even to decreased
from GM ~ S13; DTs in all PGC were decelerated in both groups, except
accelerated in Om-S13; and similarly, Cfs were decreased in GM and S9 in both
Ym and Om, whereas solely increased in S13 in both groups. In conclusion,
changes in the effects of aging and radiation exposure for HSC/PGC were significant
only in the primitive PGC (CFU-S13), in which cell cycle was depressed during
aging, and was contrarily accelerated reactively after radiation only in the primitive
PGC.
1155 CADMIUM-INDUCED TRANSFORMATION OF
HUMAN STEM CELLS.
E. Tokar and M. Waalkes. National Toxicology Program, NIEHS, Research Triangle
Park, NC.
Cadmium (Cd) is a common environmental contaminant linked to human
prostate cancer. Strong evidence indicates cancer is a stem cell (SC)-based disease,
due, in part, to the inherent resistance and indefinite proliferative potential of SCs,
allowing accumulation of sufficient oncogenic “hits” to drive neoplasia. We have
recently observed SCs possess an innate resistance and hyperadaptability to arsenic,
manifesting as a cancer SC (CSC) overproduction unique to arsenic-induced malignant
transformation but not seen with Cd-induced transformation. To determine
the reasons for this difference between these carcinogenic inorganics and
CSC dynamics, a human prostate epithelial SC line, WPE-stem, and its mature
parental cell line, RWPE-1, were chronically exposed to low-level (10 μM) Cd for
up to 10 weeks. Early exposure was highly toxic to SCs (90% toxicity at 1 week)
but, remarkably, not to mature epithelial cells. This is unusual given that SCs are
typically resistant to toxicants. The SCs rapidly recovered and were growing at a
rate similar to mature epithelial cells or control SCs by 4 weeks. Thus, it appears
Cd causes an early “bottleneck” effect specifically on SCs. Based on secreted MMP-
9 activity, a well-established marker for malignant transformation, the SCs were
potentially transformed by Cd in only 4 weeks (1500% increase in MMP-9) while
parental cells required 10 weeks of Cd exposure before indications of transformation
occurred (400% increase in MMP-9). This accelerated transformation of SCs
compared to their mature counterparts is similar to that seen with arsenic in the
same cells, indicating the stage of differentiation may be a key factor in time to acquired
malignant phenotype with both inorganics. Together, these data show that,
similar to arsenic, Cd can induce an acquired cancer phenotype in SCs and does so
much quicker than in mature cells. However, in direct contrast to arsenic, Cd selectively
kills SCs in early stages of transformation. This could account for the
quantitative difference in CSCs between these two inorganics upon acquired malignant
phenotype.
1156 GROWTH HORMONE VS. PEGYLATED GH
LIPOATROPHY IN HUMAN STEM CELL DERIVED
ADIPOCYTES CHRISTOPHER DONAHUE 1 , MARK
THIEDE 1 , SUSAN MARTIN 2 ,, JANIS VAJDOS 1 , AND
PHILLIP M. BARTHOLOMEW 3 GENETICALLY
MODIFIED MODELS CENTER OF EMPHASIS 1 ,
BIOLOGICS PHARM R&D 2 , AND DRUG SAFETY R&D 3
PFIZER R&D, GROTON, CT 06340.
P. M. Bartholomew 1 , C. Donahue 2 , M. Thiede 2 , S. Martin 3 and J. Vajdos 2 .
1 Drug Safety R&D, Pfizer Inc., Groton, CT, 2 Genetically Modified Models, Pfizer
Inc., Groton, CT and 3 Pharm R&D, Pfizer Inc., Groton, CT.
Injection site lipoatrophy occurs at a low incidence with some injectable drugs (e.g.
recombinant human growth hormone (rhGH) and insulin). In vitro, GH inhibits
lipid accumulation and stimulates lipolysis in adipocytes, a pharmacological action
consistent with lipoatrophy. Since rhGH therapy requires daily injections due to a
2-3 hr half-life, a polyethylene glycol-GH (PEG–GH) was created to provide a
weekly dosing option. While 3 month animal toxicology studies with PEG-GH
showed no evidence of lipoatrophy, it was observed at the injection site clinically.
To address the mechanism of lipoatrophy, we used a dynamic adipocyte culture differentiated
from human mesenchymal stem cells (hMSCs). We explored the concentration-dependent
action of rhGH on lipid accumulation during adipocyte formation
(Days 4-19) and maintenance (Days 19-33) as well as lipolysis of mature
adipocytes. We found GH receptor mRNA to be induced (up to 25 fold) during
differentiation and expressed throughout the 33 day culture. Addition of PEG-GH
(0.003-30 μg/ml), but not PEG, during adipocyte formation or maintenance reduced
lipid content concentration dependently, 12-36% on Day 19, 5-25% on
Day 33. In contrast, all concentrations (0.001-10 μg/ml) of rhGH reduced lipid
content (~22%) at both time points. Lipolysis measured on Day 19 by glycerol release
increased in a concentration dependent manner after 4 hour incubations with
rhGH (0.001-10 μg/ml). PEG-GH stimulated lipolysis non-dose dependently and
to a lesser degree than GH. Exposure of GH receptor-expressing human adipocytes
to GH receptor agonist results in lipid loss consistent with clinical lipoatrophy.
Thus this in vitro model appears suitable for evaluating the lipoatrophy potential of
new therapies.
1157 DIOXIN, A POTENT LIGAND OF THE ARYL
HYDROCARBON RECEPTOR, ALTERS MIGRATION OF
MURINE HEMATOPOIETIC STEM CELLS.
F. L. Casado-Pena, K. P. Singh and T. A. Gasiewicz. Environmental Medicine,
University of Rochester, Rochester, NY.
Human exposure to high levels of dioxins has been associated with increased incidence
of leukemia and lymphoma. A plausible mechanism to support these claims
has not been defined. Previously, we reported that the exposure of mice to 2,3,7,8tetrachlorodibenzo-p-dioxin
(TCDD) leads to increased relative numbers of phenotypically-defined
cell populations in bone marrow, and compromised reconstitution
of bone marrow after transplantation into irradiated host animals without
changing the absolute numbers of HSCs with short- and long-term repopulation.
AhR’s antagonists have been reported to promote the expansion of human
hematopoietic stem cells (HSC) in vitro, and HSCs from AhR-/- mice are more
proliferative. Here, we tested the hypothesis that TCDD changes the expression of
genes involved in the migratory behavior of HSCs to the bone marrow. In vivo and
in vitro migration of TCDD-treated (30 μg/kg, 7 days) HSC-enriched populations
of cells were altered by a mechanism involving phenotypic changes in CXCR4, a receptor
for a bone marrow chemotactic factor, and CD44, a receptor for molecules
present in the extra-cellular matrix of the bone marrow. Gene expression profiles
were studied at 6 and 12 hours after treatment and are consistent with a TCDD effect
on cellular trafficking of phenotypically-enriched HSCs. Our data support a
genomic-mechanism for TCDD to alter the phenotype, function and migration of
HSCs. Supported by NIH Grants ES04862, ES01247, ES016606.
1158 ARYL HYDROCARBON RECEPTOR AGONIST DIOXIN
PRODUCES ALTERATIONS IN EARLY HEMATOPOIESIS
IN MOUSE FETAL LIVER.
K. P. Singh, F. L. Casado and T. A. Gasiewicz. Environmental Medicine, University
of Rochester Medical Center, Rochester, NY.
The aryl hydrocarbon receptor (AhR) is a highly conserved, developmentally-regulated
and ligand-dependent transcription factor in the basic helix-loop-helix superfamily
of DNA-binding proteins. The 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD; Dioxin) is the most potent ligand for AhR known, and it is used to study
SOT 2011 ANNUAL MEETING 247