The Toxicologist - Society of Toxicology

larly suppressed LPS-induced TNF-α production both in primary human cultures
as well in THP-1 cell line, while IL-8 was suppressed in THP-1, unaffected in female
leukocytes and increased in male leukocytes. In THP-1 cells, we could
demonstrate that the effect was pre-transcriptional, as assessed by a reduction in
both LPS-induced TNF-α and IL-8 mRNA expression. A different effect of PFOA
and PFOS on LPS-induced NF-kB activation could be demonstrated, suggesting a
different mode of action for these PFCs. Overall, the studies suggest that PFCs directly
suppress cytokine secretion in THP-1 cells. Among the different PFCs,
PFOA appears to be the less effective compound. Acknowledgements: This research
was supported in part by the Intramural Research Program of the National Institute
of Environmental Health Sciences, National Institutes of Health.
1556 TH2 SKEWING BY NRF2 ACTIVATION IN CD4+ T
CELLS AS EVIDENCED BY INCREASED PRODUCTION
OF IL-4, IL-5, AND IL-13 AND DECREASED
PRODUCTION OF IFNγ
C. E. Rockwell 1 , P. E. Fields 2 and C. D. Klaassen 1 . 1 Department of Pharmacology,
Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS
and 2 Department of Pathology & Laboratory Medicine, University of Kansas Medical
Center, Kansas City, KS.
Nuclear factor erythroid 2 related factor 2 (Nrf2) is a transcription factor that is activated
by cellular stress, including oxidative and electrophilic stresses. Numerous
compounds are used experimentally as Nrf2 activators, including tBHQ and BHA.
Interestingly, tBHQ and BHA are also used commercially as food preservatives.
Nrf2 has been reported to have anti-inflammatory activity in various models of inflammation,
including septic shock and others. Whereas Nrf2 has been characterized
in macrophages, much less is known about the role of Nrf2 in T cells.
Accordingly, the purpose of the present studies was to ascertain the function of
Nrf2 in T cells. Our studies demonstrate that tBHQ and BHA suppress IFNγ production
by CD3/CD28-activated splenocytes. The decrease in IFNγ transcription
by Nrf2 correlates with decreased binding to the AP-1 and NFκB consensus binding
sites as well as decreased nuclear accumulation of c-fos, JunD and p65 (NFκB).
To determine whether inhibition of IFNγ production by splenocytes activated with
a T cell-specific activator is a cell-autonomous effect within T cells or whether other
cell types are involved, the effect of Nrf2 activators on cytokine production by isolated
CD4+ cells was assessed. tBHQ suppressed production of IFNγ, a TH1 cytokine,
in CD4+ cells derived from wild-type, but not Nrf2-null mice. Conversely,
tBHQ stimulated production of the TH2 cytokines, IL-4, IL-5, and IL-13 in a
Nrf2-dependent manner. In contrast to the TH1/TH2 cytokines, Nrf2 had little
effect on production of IL-10 and TNFα. Collectively, the data suggest that tBHQ
induces skewing toward the TH2 subtype through activation of Nrf2. Further studies
will be needed to determine whether the addition of tBHQ to foods as a preservative
may promote food allergy and/or compromise cell-mediated immunity.
(Supported by NIH grants DK081461, ES07079, and RR021940.)
1557 5-NITROAPOCYNIN REVERSES LPS-, BUT NOT PMA-
INDUCED IL-6 RELEASE BY MOUSE J774
MACROPHAGES: A POSSIBLE ROLE OF PROTEIN
KINASE-C.
A. C. Raghavamenon 1 , S. Babu 1 , V. Achuthan 1 , X. Gao 1 , O. D’Auvergne 2 , R.
Atmakuru 3 and R. M. Uppu 1 . 1 Environmental Toxicology, Southern University and
A&M College, Baton Rouge, LA, 2 Biological Sciences, Southern University and A&M
College, Baton Rouge, LA and 3 Analytical Chemistry, International Institute of
Biotechnology and Toxicology, Padappai, Tamil Nadu, India.
Recently we have shown that apocynin (Apo) can undergo metabolic transformation
to 5-nitropaocynin (5-NitroApo) by perynitrite/CO 2
. In the present study we
examined the effects of 5-NitroApo and Apo on the release of inflammatory cytokines,
IL-6 and TNFα by mouse J774 macrophages. Pre-incubation of
macrophages with 50 μM 5-NitroApo for I h reduced the lipopolysacharide
(LPS;10 μM)-induced IL-6 release in the culture supernatants. Exposure to Apo
also decreased the IL-6 secretion but the magnitude of this decrease was smaller
than that observed with 5-NitroApo. In these experiments, 5-NitroApo was not cytotoxic
to macrophages in the concentration range of 0 to 100 μM for 24 h.
Phorbol myristate acetate (PMA), on the other hand, was somewhat cytotoxic to
macrophages and this could not be reversed by either Apo or 5-NitroApo. In assays
using PMA, 5-NitroApo but not Apo increased the release of IL-6. In both LPS
and PMA exposures, macrophages showed little or no increase in the release of
TNFα. Apo and 5-NitropApo, which did not alter the PMA- or LPS-induced release
of TNFα, per se lowered the secretion of TNFα by macrophages. These observations
suggested that Apo and 5-NitroApo inhibit the LPS-induced NFKB activation
(presumably mediated via Toll-like receptor 4, TLR4) and thereby reduces the
associated inflammatory response measured in terms of IL-6 release. On the other
hand, the inflammatory response by PMA, mediated through activation of protein
kinase-C (PKC), could be increased by 5-NitoApo. [Funding support from NIH
(P20 RR16456) and Department of Education (PO31B040030) is acknowledged.
1558 TUNGSTEN INDUCES DNA DAMAGE AND ALTERS
GROWTH OF DEVELOPING B LYMPHOCYTES.
C. Guilbert and K. K. Mann. Lady Davis Institute for Medical Research, McGill
University, Montréal, QC, Canada.
Tungsten is used widely in modern life in everything from household goods to technologically
advanced material. However, little is known about the consequences of
tungsten exposure. The lack of information concerning the toxicities associated
with tungsten has been highlighted recently when high tungsten levels were discovered
at the sites of several pediatric leukemia clusters. In these clusters, the majority
of the children developed acute lymphoblastoid leukemia of the preB lymphocyte
subtype. Thus, we investigated whether tungsten exposure alters the growth of preB
cells and defined signaling pathways induced by tungsten. We exposed a co-culture
system of the BU-11 preB cell line grown on BMS2 stromal cells to tungsten at
doses found at the sites of the leukemia clusters. Growth, cell cycle, and apoptosis
were determined in both cell types. While no significant changes in any of these parameters
were observed in the BMS2 stromal cells, tungsten treatment for 48 hours
decreased the BU-11 cell number, which correlated with an accumulation of cells in
G0/G1 and increased apoptosis. Unlike polycyclic aromatic hydrocarbons, tungsten
induced apoptosis in BU-11 cells grown in rIL-7 and therefore, is independent
of the stromal cell layer. These results were confirmed in primary murine bone marrow
cultures and in human peripheral blood mononuclear cells. In order to begin
analysis of signaling pathways induced by tungsten, cDNA microarrays were performed
in a leukemic cell line following 24 hours of tungsten treatment and integrated
pathway analyses were performed. Genes involved in DNA damage and ER
stress were highly upregulated following tungsten exposure and validated by qPCR.
An increase in DNA damage was confirmed by COMET assay. These results suggest
that the developing B lymphocyte population is sensitive to tungsten-induced
toxicities and that tungsten exposure may contribute to leukemogenesis.
1559 TRANSCRIPTIONAL REGULATION OF
HISTOCOMPATIBILITY COMPLEX CLASS II (MHC-II)
GENES BY AGONIST AND ANTAGONIST OF
PREGNANE X RECEPTOR.
E. Fuentes-Mattei 1, 2 , R. I. Rodriguez-Cotto 1, 2 and B. D. Jimenez-Velez 1, 2 .
1
Biochemistry, UPR Medical Sciences Campus, San Juan and 2 Center for
Environmental and Toxicological Research, UPR Medical Sciences Campus, San Juan.
Pregnane X Receptor (PXR) is a sensor to a broad range of natural and synthetic
xenobiotics to mediate the induction of CYP3A and other drug metabolizing enzymes.
Accumulating evidence suggests the role of PXR in other transcriptional
regulations (e.g., energy and glucose metabolism, lipid metabolism and inflammation).
Adding to these findings, activation of rodent PXR induces the Major
Histocompatibility Complex class II (MHC-II) in primary hepatocytes. The aim of
this study is to investigate further the role of PXR and its possible involvement in
the immune response. First, we determined the promoter activity of MHC-II
(HLA-DRα) experiments with or without over-expression of human PXR in cotransfected
HT-1080 fibrosarcoma cells. Using human bronchial epithelial cells
(BEAS-2B) we measure mRNA levels of MHC-II after treatments with PXR agonist
at different time intervals. To determine a possible role of PXR in the IFNγ
pathway, we also used a PXR antagonist (sulforaphane) to evaluate changes in the
IFNγ-mediated transcriptional regulation of HLA-DRα. HLA-DRα promoter activity
was induced by activation of endogenous PXR and by activation of the IFNγ
pathway in co-transfected HT-1080. Over-expression of PXR in co-transected HT-
1080 cells also induced the HLA-DRα promoter activity. In BEAS-2B cells, both
HLA-DRα and PXR mRNAs were elevated after treatment with rifampicin or
IFNγ. Other PXR agonists, SR12813 and dexamethasone, also up-regulated the
HLA-DRα mRNA levels. The PXR antagonist, sulforaphane, inhibited the IFNγmediated
transcriptional up-regulation of HLA-DRα after 4h and 24h in BEAS-
2B. These findings support the hypothesis of a potential involvement of PXR enhancing
transcriptional induction of MHC-II genes. These results continue to add
to possible associations between immune and detoxification regulations through
PXR as means of a xeno-protective mechanism.
SOT 2010 ANNUAL MEETING 331