The Toxicologist - Society of Toxicology

1135 BACH2 BINDING TO Prdm1 AS A MECHANISM OF 2, 3,
7, 8-TETRACHLORODIBENZO-P-DIOXIN(TCDD)-
MEDIATED IMMUNE SUPPRESSION.
A. S. Phadnis 1, 3 , K. DeAbrew 4 , R. S. Thomas 4 and N. E. Kaminski 2, 3 . 1 Genetics
Program, Michigan State University, East Lansing, MI, 2 Pharmacology & Toxicology,
Michigan State University, East Lansing, MI, 3 Center for Integrative Toxicology,
Michigan State University, East Lansing, MI and 4 Hamner Institutes for Health
Sciences, Research Triangle Park, NC.
Altered B cell differentiation and decreased IgM production are some of the major
AHR-dependent effects of TCDD seen both in vitro and in vivo. Previously, using
a genome-wide analysis of ligand-dependent AHR DNA binding, we identified
Bach2 as a novel gene directly regulated by the AHR in a murine B lymphoma cell
line CH12.LX. Bach2 is a B cell specific repressor of Prdm1 and mediates its repressive
functions by binding to Maf recognition elements (MAREs) in the regulatory
regions of Prdm1. qRT-PCR experiments performed in resting and LPS-activated
CH12.LX cells showed induction of Bach2 mRNA levels at 2h in presence of
TCDD; concordant with the chromatin immunoprecipitation and gene expression
microarray experiments. To further elucidate the role of Bach2 in regulating Prdm1,
electrophoretic mobility shift assays were performed on nuclear extracts from LPSactivated
and resting CH12.LX cells treated with TCDD. In both Prdm1 promoter
and intron 5 MAREs, increased TCDD-inducible DNA binding activity of Bach2
was seen at 4h in LPS-activated cells as compared to treatment with LPS or TCDD
alone. A TCDD-concentration dependent DNA binding activity was also observed
at the intron5 MARE. In contrast, maximum DNA-binding was seen at 0.1nM
TCDD in the promoter MARE at 4h post-treatment. In order to identify the
MARE- binding protein complex, supershift assays were performed using an anti-
Bach2 antibody and non-specific goat IgG. Specific Bach2 binding was observed at
the intron5 MARE but not at the promoter MARE. These results suggest differential
binding activity of Bach2 to the two MARE elements in presence of TCDD.
Taken together these findings shed light on a new mechanism of controlling Prdm1
in TCDD-mediated immune suppression. (Supported by NIH ES04911 and
ES02520)
1136 DIDOX (DIHYDROXYBENZOHYDROXAMIC ACID)
AMELIORATES MACROPHAGE AHR ACTIVATION,
CYP1A1/AHR INDUCTION, AND OXIDATIVE STRESS
PROFILES INDUCED BY PCB-126.
C. D. Rice, V. S. Gallichio and T. M. Matsebatlela. Biological Sciences, Clemson
University, Clemson, SC.
Exposure to AhR-activating xenobiotics is associated with oxidative stress and inflammation.
For example, PCB-126 is a potent and well-studied AhR-ligand and
inducer of CYP1A1/2 and Phase II metabolic enzymes. Intracellular ROI increases
in cells within a few hrs of exposure, long before mRNA transcription and protein
induction. Thereafter, levels of ROI continually rise for several days due to a high
affinity of PCB-126 for the AhR and recalcitrant metabolism. After a 24 hr exposure
of RAW264.7 murine macrophages to 1 μM PCB-126 we show (using qRT
PCR) that expression levels of GSH-peroxidase and SOD genes are suppressed,
while expression levels of of NADPH-oxidase genes are elevated. As expected,
CYP1A1 gene/protein expression is markedly induced and associated with elevated
superoxide anion and H2O2 production. In search of strong, but non-toxic antioxidants
that may ameliorate AhR-associated ROI and inflammation, we found that
Didox (3,4-Dihydroxybenzohydroxamic acid) is very potent. Several GSH-peroxidase
genes and GSH reductase, peroxiredoxins, thioredoxin reductase, all three
types of SODs, and catalase are elevated by Didox. Furthermore, Didox reverses oxidative
stress gene profiles initiated by PCB-126. Of particular note, Didox mimics
LPS in that exposure suppresses the expression of both CYP1A1 and AhR genes.
We postulate that Didox may have significant potential in ameliorating the toxicity
of several classes of AhR-activating immunotoxic compounds.
1137 MECHANISTIC INVESTIGATION OF TCDD EFFECTS
ON CD40 LIGAND-INDUCED ACTIVATION OF
PRIMARY HUMAN B CELLS: PERTURBATION OF
IMMEDIATE AND PERSISTENT SIGNALING.
H. Lu 1, 2 , R. Crawford 1, 2 and N. E. Kaminski 1, 2 . 1 Center for Integrative Toxicology,
Michigan State University, East Lansing, MI and 2 Pharmacology & Toxicology,
Michigan State University, East Lansing, MI.
The B cell is well established as a sensitive cellular target of 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) in suppression of the primary antibody response
in mice. However, data concerning TCDD effects on human B cells remains scarce.
Previous findings that the CD40 ligand-induced IgM response in primary human
B cells was decreased by TCDD led us to investigate the mechanisms by which
TCDD impairs human B cell effector function using the same in vitro activation
model. Critical steps involved in the progression of a resting B cell to a differentiated
antibody secreting cells, namely activation, proliferation, and transcriptional
regulation of plasmacytic differentiation were assessed using real-time PCR and
flow cytometry analyses. TCDD exhibited a modest effect on proliferation, but
profoundly attenuated the activation of human B cells, as evidenced by decreased
expression of surface activation markers CD80, CD86, and CD69, which was consistently
observed among multiple human donors. The impaired activation, which
correlated with decreased cell viability, prevented B cells from progressing toward
the stage of plasmacytic differentiation. Phosflow techniques were applied to investigate
the mechanisms for impairment of B cell activation, and showed that TCDD
produced perturbation of immediate and/or persistent activation of mitogen-activated
protein kinases, protein kinase B/Akt, signal transducer and activator of transcription
1, 3 and 5, AP-1/c-Jun, and RelA/p65 in activated human B cells.
Collectively, this series of study demonstrates that CD40L-induced activation of
human B cells is impaired by TCDD, through mechanisms that involve the perturbation
of multiple signaling cascades downstream of CD40 and the cytokine receptors.
Supported in part by NIH P42 ES04911 and ES02520, the Dow Chemical
Company, and a research fellowship from Syngenta AG.
1138 AH RECEPTOR MEDIATED RESTORATION OF SPLEEN
AND BONE MARROW FOLLOWING THE CYP1B1-
DEPENDENT ADVERSE EFFECTS OF POLYCYCLIC
AROMATIC HYDROCARBONS (PAHS).
A. U. N’jai 1, 3 , M. Larsen 2 , C. R. Jefcoate 2, 3 and C. J. Czuprynski 1, 3, 4 .
1 Pathobiological Sciences, University of Wisconsin, Madison, WI, 2 Pharmacology,
University of Wisconsin, Madison, WI, 3 METC, University of Wisconsin, Madison,
WI and 4 Food Research Institute, University of Wisconsin, Madison, WI.
Bone marrow (BM) lymphoid progenitors emigrate to the spleen and thymus for
maturation into effector B and T cells, respectively. We have previously reported
rapid in vivo suppression of BM progenitors by exposure to certain PAHs. Here, we
assess in a coordinated manner the effects of PAHs on lymphoid populations in the
bone marrow, blood, spleen and thymus. IP administration of 7,12-dimethylbenz(a)anthracene
(DMBA) rapidly suppressed (>50% at 6h) lymphoid CFU-preB
progenitor cell activity. By 48 h, cell numbers in the BM, spleen and thymus exhibited
similar decreases. Oral administration of DMBA, which results in its clearance
within 12h, decreased progenitor cell activity at 6 h but this returned to normal by
48h. IP administration of benzo(a)pyrene (BP) had an adverse effect on BM progenitors
that was comparable to DMBA at 6 h. However, this was followed by an
Ah Receptor-dependent recovery of progenitor cell activity. By contrast, similar
suppression by each PAH was seen for total blood lymphocytes (6h) and thymocytes
(48h). The effects of DMBA and BP were both dependent on Cyp1b1 metabolism.
BM, spleen, thymus, and blood lymphoid cell populations generally declined
and recovered in a temporal pattern similar to the changes noted for BM
progenitor cells. These observations are consistent with replenishment of the blood,
spleen and thymus populations subsequent to restoration of BM progenitors.
Paradoxically, gene expression responses to DMBA were almost absent in BM cells
after 12h but were extensive for BP. We infer that Cyp1b1 mediated DMBA metabolism
specifically affects the few BM progenitor cells. Many BP gene responses
were AhR dependent and may be link to progenitor recovery.
1139 ACTIVATION OF THE ARYL HYDROCARBON
RECEPTOR BY TCDD SUPPRESSES SENSITIZATION IN
A MOUSE PEANUT ALLERGY MODEL.
V. J. Schulz 1 , J. Smit 1, 2 , L. Boon 3 , M. van den Berg 1 , M. van Duursen 1 and R.
Pieters 1 . 1 Immunotoxicology, Institute of Risk Assessment Sciences, Utrecht University,
Utrecht, Netherlands, 2 Utrecht Center for Food Allergy, Utrecht, Netherlands and
3 Bioceros B.V., Utrecht, Netherlands.
Food allergy is an increasing health problem in Western countries. Previously, it was
shown that the intensity of food allergic reactions can be regulated by regulatory T
(Treg) cells. In addition, it was shown that activation of the aryl hydrocarbon receptor
(AhR) regulates T cells responses and induces Treg cells. Therefore, we investigated
whether activation of the AhR could suppress food allergic responses
through the induction of Treg cells. C3H/HeOuJ mice received 2,3,7,8-tetrachlorodibenzo-p-dioxin
TCDD (0.6, 1.7, 5 and 15 μg/kg BW) orally and were sensitized
to peanut by administering peanut extract (PE) by gavage in the presence of
cholera toxin (CT). We found that activation of the AhR by TCDD dose-dependently
decreased PE-specific IgE, IgG1 and IgG2a. Furthermore, PE-induced IL-5,
IL-10 and IL-13 were decreased by TCDD, whereas IFN-γ production was increased.
The percentage of Treg cells (CD4+CD25+Foxp3+) increased dose-dependently
by TCDD treatment in both spleen and MLN. However, the absolute
SOT 2011 ANNUAL MEETING 243