The Toxicologist - Society of Toxicology

1168 DIFFERENTIAL PAH EFFECTS MEDIATED BY CYP1A1
AND CYP1B1 IN THE BONE MARROW SPECIFICALLY
TARGET HEMATOPOIETIC PROGENITORS.
M. L. Larsen 1 , A. U. N’Jai 3 , C. J. Czuprynski 3, 2 and C. R. Jefcoate 1, 2 .
1 Pharmacology, University of Wisconsin, Madison, WI, 2 Molecular and
Environmental Toxicology Center, University of Wisconsin, Madison, WI and 3 School
of Veterinary Medicine, University of Wisconsin, Madison, WI.
The polycyclic aromatic hydrocarbons (PAHs), benzo(a)pyrene (BP) and 7,12-dimethylbenzanthracene
(DMBA), produce very different effects in a Min-mouse
cancer model and differential suppression of hematopoietic progenitors in mouse
bone marrow (BM). In each model, DMBA is much more active than BP and the
effects are dependent on Cyp1b1. Colony forming unit assays show that DMBA
suppresses lymphoid progenitor activity within 6 h. BP is much less effective, due
to an Ah receptor (AhR)-mediated recovery process. Microarray analyses show few
DMBA responses, but numerous BP responses (25 versus 600). Nine gene responses,
which were similar for each PAH and for TCDD, are attributed to direct
AhR stimulation. These include Cyp1a1, Cyp1b1, and AhRR. Many BP responses,
like the recovery process, were AhR dependent, but resistant to DMBA. The time
dependence of BP-responses demonstrated an inflammatory group that responded
within 6 h (chemokines, cytokines, COX-2) and decayed within 24 h, and a second
group (Nqo1 muGSTs), which showed the inverse response. Gene expression
changes in BM of Cyp1b1-/- mice demonstrated the substantial altered expression
of many developmental regulators. Remarkably, inflammatory gene responses to
DMBA, which were absent in WT mice, were increased to BP levels. Thus, removal
of BM Cyp1b1 metabolism appears to enhance local DMBA responses. In Cyp1a1-
/- mice, many BP-inflammatory responses declined and a cluster of other gene responses
increased. We hypothesize that Cyp1a1 generates BP quinones in BM that
initiate selective oxidative stress responses that contribute to protection, while endogenous
Cyp1b1 metabolites control developmental processes, consistent with a
role in embryogenesis.
1169 IMMUNOMODULATORY RESPONSE OF MOUSE BONE
MARROW-DERIVED MESENCHYMAL STROMAL
CELLS (MBMSCS) TO STIMULATION WITH
LIPOPOLYSACCHARIDES (LPS).
N. V. Gorbunov, B. R. Garrison, G. Ledney and J. G. Kiang. Radiation Combined
Injury Program, Armed Forces Radiobiology Research Institute, Bethesda, MD.
Mortality induced by gram-negative bacterial sepsis remains a serious problem of
complicated injury due to penetrating wounds, burns, blunt trauma, radiation,
chemical exposures, or combinations thereof. A promising therapeutic tool for
management of trauma and sepsis is transfusion with bone marrow-derived mesenchymal
stem cells (BMSCs) that exhibit immunomodulatory capacity. However,
the molecular mechanisms underlying BMSC action in septic conditions are poorly
understood. Thus, this report focuses on in vitro investigations of the response of
mouse BMSCs (mBMSCs) to stimulation with products of gram-negative bacteria,
LPS. mBMSCs were obtained from the femur bone marrow of B6D2F1/J female
mice. The cells were expanded and cultivated in hypoxic conditions for 28 days in
MESENCULT medium. The cell phenotype, proliferative and colony-forming activity
were analyzed with flow cytometry and immunofluorescence imaging; the
cells were identified by BMSC-positive markers: CD44, STRO1, SCA1. mBMSCs
were exposed to 0.05 - 2.5 μg/ml LPS for 1-3 h and analyzed at different timepoints.
LPS-induced gene and protein expression of immunomodulatory effectors
(IL-1, IL-6, IL-8, and iNOS) were determined by qRT-PCR and immunoblotting
techniques. Nuclear translocation of p65-NFkB and nitric oxide (NO) production
in the cells was assessed with fluorescent imaging. Treatment of mBMSCs with LPS
resulted in a substantial increase in gene expression of IL-1α, IL-1β, and iNOS that
occurred in a dose- and time-dependent manner. At 24 h following exposure to 0.5
μg/ml LPS, expression of NFkB, IL-1β, and iNOS proteins, and NO production
in the cells was increased 2-, 12-, 10-, and 14-fold, respectively. These data are consistent
with the idea that LPS-induced alterations in mBMSCs are mediated by the
Toll-like receptor 4/NFkB axes that, in turn, modultate the host adaptive responses
during septic conditions. (Supported by NIH/NIAID YI-AI-5045-04).
1170 FINE PARTICULATE MATTER (PM 2.5 ) EXPOSURE
AFFECTS CIRCULATING AND BONE MARROW
ENDOTHELIAL PROGENITOR CELLS.
P. Haberzettl, J. Lee, D. Duggineni, J. McCracken, A. Bhatnagar and D. J.
Conklin. Diabetes and Obesity Center, University of Louisville, Louisville, KY.
Inhalation of fine particulate matter (PM 2.5 ) induces endothelial dysfunction and
increases the risk for cardiovascular disease and mortality. Endothelial progenitor
cells (EPC) contribute to endothelium health and we reported that exposure to am-
250 SOT 2011 ANNUAL MEETING
bient or concentrated ambient PM 2.5 (CAPs) decreases the number of circulating
EPC in humans and mice. Thus, we performed experiments to assess effects of
PM 2.5 exposure on the recruitment/mobilization of EPC. Mice (male; C57BL/6;
12-wks) were exposed up to 30 consecutive days (6h/day) to HEPA-filtered air or
CAPs (≈8-fold conc.; 80-100 μg/m 3 ) of urban Louisville air using a versatile aerosol
concentration enrichment system (VACES). CAPs exposure for 4, 9 or 30 days significantly
decreased the number of circulating EPC measured as Flk-1 + /Sca-1 + -cells
by FACS by 32.6±13.8, 35.8±10.1 or 46±16.9 (% control), respectively. EPC levels
returned to baseline within 1 week following a 9day CAPs exposure, indicating
a reversible PM effect. CAPs exposure (9-day) significantly increased bone marrowderived
Flk-1 + /Sca-1 + -cells by 1.75±0.1-fold when compared with controls.
Similarly, CAPs exposure (9-day) significantly increased colony forming units
(1.6±0.1-fold) and the number of Flk-1 + /Sca-1 + or acLDL + /UE-lectin + cells (1.8 ±
0.1 or 1.6 ± 0.1 fold) of bone marrow-derived cells in culture compared with controls.
These results indicated that PM blocked EPC mobilization. To test this, mice
exposed for 9 days to air or CAPs were subjected to an EPC mobilization protocol
(VEGF, 100μg/kg/d * 4d and AMD3100, 5mg/kg) and blood levels of EPC were
analyzed. VEGF/AMD3100 treatment doubled the number of circulating Flk-
1 + /Sca-1 + -cells in air controls but not in CAPs-exposed mice (-48±14 % EPC).
These results indicate that inhalation of PM 2.5 affects EPC mobilization leading to
suppressed blood EPC level, which implicates EPC as a potential mechanistic link
between PM 2.5 exposure and CVD.
1171 ALDH1B1 IS A MARKER FOR HUMAN COLON
CANCER.
Y. Chen 1 , A. Matsumoto 1 , D. Orlicky 2 , S. Singh 1 and V. Vasiliou 1 .
1 Pharmaceutical Sciences, University of Colorado Denver, Aurora, CO and 2 Pathology,
University of Colorado Denver, Aurora, CO.
Aldehyde dehydrogenases (ALDHs) belong to a superfamily of NAD(P) + -dependent
enzymes, which catalyze the oxidation of various endogenous and exogenous
aldehydes to their corresponding acids. Increased expression and/or activity of
ALDHs, particularly ALDH1A1, have been reported to occur in human cancers of
various origins. It is proposed that ALDH1A1 may play a critical role in cancer
stem cells due to its high affinity for the oxidation of retinal. Nevertheless, the identity
of ALDH isozymes in contributing to the enhanced ALDH activity in a specific
type of human cancers remains to be elucidated. ALDH1B1 has recently been
characterized as a mitochondrial ALDH that actively metabolizes acetaldehyde. In
this study, we examined the expression of ALDH1A1 and ALDH1B1 in human tumors
of colon, lung, breast and ovary using tissue array (NIH) by immunohistochemistry.
We observed a much higher percentage of immunopositivity for
ALDH1B1 (>60%) in these cancerous tissues than that for ALDH1A1 ( ALDH1B1 in ovary
> ALDH1B1 in lung or breast > ALDH1A1 in lung > ALDH1A1 in colon or ovary
> ALDH1A1 in breast. This result demonstrates that ALDH1B1 isozyme is more
profoundly expressed in studied human cancer tissues than ALDH1A1, particularly
in colon cancer. We further examined the mRNA and protein levels of the two
ALDHs in six human colon cancer cell lines. ALDH1A1 was found to be abundant
in three cell lines, whereas ALDH1B1 was highly expressed in all six cell lines. In
conclusion, our study suggests that ALDH1B1 is preferably upregulated in human
colon cancer tissues and cell lines, making it a potential marker for human colon
cancer. Supported in part by NIH grant R21AA017754-01.
1172 DIFFERENTIAL TOXICITIES OF KINASE INHIBITORS
(KI) ON BONE MARROW PROGENITORS FROM
DIFFERENT SPECIES.
E. Clarke and G. dos Santos. ReachBio, Seattle, WA. Sponsor: R. Steigerwalt.
KIs represent a new class of rationally designed drugs. The success of the initial
therapeutic KIs in treating leukemia has spurred the development of new KIs for
treating various other cancers and inflammation. However, since myelotoxicity is
often associated with administering these agents, and as the use of KIs extends to
non-oncology applications, there is a need to develop compounds with lower levels
of toxicity than previously associated with this drug class. We previously showed
that the IC50 value for 6 KIs using the human colony-forming unit-granulocyte
macrophage (CFU-GM) assay correlated with clinical neutropenia and could be
used to accurately rank the myelotoxic potential of compounds to other members
of the same chemical class. Interestingly, studies have revealed significant differences
between human, dog, rat and mouse CFU-GM sensitivities to certain pharmaceuticals.
Since multiple animal models are often used in toxicity testing, we