The Toxicologist - Society of Toxicology

and cytotoxic effects; however, its effect on dioxin-induced level of CYP1A1 has
not been studied before. Therefore, the aim of this work is to study the effect of
harmine and its main metabolite, harmol, on dioxin-induced AhR-mediated signal
transduction both in mouse and human hepatoma cell lines. For this purpose;
HepG2 and Hepa 1c1c7 cells were incubated with increasing concentrations of
harmine and harmol (0.5-12.5 μM) in the presence of dioxin (1nM). Our results
showed that harmine and harmol significantly inhibited the dioxin-induced
CYP1A1 at mRNA, protein and activity levels, in a concentration-dependent manner,
in human hepatoma HepG2 cells. However, the effect of harmine was more
pronounced. Moreover, harmine and harmol inhibited the induction of CYP1A1
activity induced by two other AhR ligands, namely, 3-methylcholanthrene and βnaphthoflavone.
The ability of harmine to affect the induced level of CYP1A1 was
strongly correlated with its ability to reduce the AhR-dependent luciferase activity
and the electrophoretic mobility shift assay (EMSA). At posttranslational level,
both harmine and harmol decreased CYP1A1 protein stability, suggesting that
posttranslational mechanism is involved. Furthermore, harmine and harmol inhibited
the induction of Cyp1a1 caused by dioxin in mouse hepatoma Hepa 1c1c7
cells. We concluded that harmine and harmol can modulate the dioxin-induced
CYP1A1 level at transcriptional and posttranslational levels. Furthermore, these
data may represent novel mechanisms by which harmine and its metabolite, harmol,
interfere with the dioxin-mediated toxic effects including carcinogenicity.
Acknowledgement: This work was supported by Natural Sciences and Engineering
Research Council of Canada.
406 E7 VIRAL ONCOPROTEIN NEGATIVELY INFLUENCES
THE TRANSCRIPTIONAL ACTIVITY OF THE L1MD
RETROTRANSPOSON PROMOTER UNDER
CONDITIONS OF CELLULAR STRESS.
D. E. Montoya-Durango 1, 2 and K. S. Ramos 1, 2 . 1 Biochemistry and Molecular
Biology, University of Louisville, Louisville, KY and 2 Center for Genetics and
Molecular Medicine, University of Louisville, Louisville, KY.
LINE-1 (L1) retrotransposons are mobile elements that modify the eukaryotic
genome by a copy and paste mechanism that allows them to reinsert elsewhere in
the genome to cause disease. L1 activation is observed under conditions of chemical
and oxidative stress. We previously identified mouse L1Md-A5 as a redox-regulated
genetic element and found both antioxidant and E2F/Rb-binding sites within
the 5’ promoter region that confer transcriptional activation and repression, respectively.
The viral oncoprotein E7 inactivates Rb protein and associates with basal
transcription factors including the AP1 family members c-Jun, JunB, JunD, and c-
Fos. Since Rb recruits histone deacetylase (HDAC) activity to repress L1 transcription,
we investigated (i) the effects of E7 and Rb complex on the mouse L1 5’UTR
and (ii) the changes in protein complex formation in primary cells under redox
stress conditions. Transient transcription assays in HeLa cells were employed to
monitor the effect of forced E7, Rb, and HDAC2 expression on L1MdA-A5 promoter
activity following challenge with the carcinogen benzo(a)pyrene (BaP). E7
ablated the response of the reporter gene to carcinogen treatment, but did not interfere
with basal transcriptional activity. Rb overexpression transactivated the promoter,
but this effect was quenched when E7 alone or in combination with
HDAC2 were added. Using gel filtration chromatography we found that in primary
vascular smooth muscle cells subjected to BaP treatment, Nrf2 and JunD proteins
shift to a macromolecular complex of 1 MDa, while JunD complexes between
700-100 kDa disappear. We conclude that E7 associates with transcription factors
required for proper assembly of the transcription macromolecular complexes that
assemble on the ARE and E2F/Rb. This is the first report showing that E7 might be
involved in the regulation of L1 retroelement activity via both the ARE and
E2F/Rb-binding sites.
407 NEOPLASTIC LUNG CELL PROLIFERATION,
STIMULATED BY ALVEOLAR MACROPHAGE-DERIVED
IGF-1, CAN BE ABROGATED BY THE COMBINED
INHIBITION OF MEK AND PI3K.
J. M. Fritz, L. D. Dwyer-Nield and A. M. Malkinson. Department of
Pharmaceutical Sciences, University of Colorado Denver, Aurora, CO.
Rationale: In human non-small cell lung cancer, chronic inflammation resulting
from toxin exposure or underlying pathology greatly promotes the growth of initiated
cells. In mouse models, tumor growth is enhanced by chemically-induced inflammation.
This chronically increases alveolar macrophage infiltration, paralleling
that which occurs naturally later in lung tumor progression. These observations
suggest that macrophage recruitment stimulates neoplastic proliferation. Herein,
we show that macrophage-derived IGF-1 is one factor that significantly promotes
tumor growth. Methods: Macrophage conditioned media (MØCM) was generated
from freshly isolated mouse bronchoalveolar lavage (BAL) cells. Macrophages were
also co-cultured with primary murine tumor cell isolates or stable neoplastic lung
epithelial cell lines in vitro. Results: MØCM and macrophage co-culture stimulate
tumor cell proliferation to similar degrees. IGF-1 is highly abundant in BAL fluid,
compared to other cytokines. IGF-1 levels are 3.5x higher in tumor-bearing lungs
vs. naïve, and tumor-educated BAL macrophages produce 2x more. Recombinant
IGF-1 stimulates neoplastic growth in vitro and is additive to MØCM, while other
factors such as IL-1β and EGF had no growth effect. Decreasing MØCM IGF-1
levels by siRNA or immuno-depletion reduces the growth stimulation in subsequently
treated cells. MØCM and IGF-1 increase Erk and Akt activation in neoplastic
cells, resulting in increased cyclin D1 expression and increased proliferation.
Combined pharmacologic inhibition of both MEK and PI3K blocks the effects of
MØCM and IGF-1, unexpectedly increasing Akt activity, but decreasing cyclin D1
expression consistent with suppressed neoplastic proliferation. Conclusions:
Inhibition of MEK and PI3K ablates macrophage-derived IGF-1-induced neoplastic
lung cell proliferation but induces hyper-activation of Akt. (Supported by
CA132552; Fritz, J.M. is an AFPE Fellow)
408 MASS SPECTROMETRY TECHNIQUES TO STUDY
PROTEIN-LIGAND INTERACTIONS AND MOLECULAR
TOXICOLOGY PATHWAYS.
K. E. Yamada 1, 2 , C. M. Ryan 3 , J. P. Whitelegge 3 and C. D. Eckhert 1, 2 .
1 Molecular Toxicology IDP, University of California Los Angeles, Los Angeles, CA,
2 Environmental Health Sciences, University of California Los Angeles, Los Angeles, CA
and 3 The Pasarow Mass Spectrometry Laboratory, The NPI-Semel Institute, David
Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA.
Native protein mass spectrometry is an under utilized technique to study proteinprotein
and protein-ligand interactions. Notable advantages of this method are the
ability to study direct binding and the ability to detect molecules without any labeling.
Traditional binding experiments usually depend on radio, immuno, or fluorescent
labels and mass spectrometry circumvents this requirement by direct mass
measurement. Furthermore, identification of specific binding site is possible. Here
we look at the effects of boric acid on the interaction of FKBP12 with its endogenous
ligand, cADPR. This technique has implications in understanding toxicity
mechanisms and pathways on the molecular level. It is sensitive enough to detect
picogram quantities of protein and resolve mass differences of less than 1 dalton.
Furthermore, advances in spraying conditions allow us to study the effects of ligands
on protein conformational changes. Mass spectrometry has the potential to be
a very powerful tool for molecular toxicologists.
409 DICLOFENAC INHIBITS TNFα-INDUCED NF-κB
NUCLEAR SHUTTLING CAUSING SYNERGISTIC
HEPATOCYTE APOPTOSIS VIA CASPASE-8.
L. Fredriksson, B. Herpers, Z. Di and B. van de Water. Toxicology, LACDR,
Leiden University, Leiden, Netherlands.
Drug-induced liver injuries (DILIs) are the major cause of drug failures and are
often idiosyncratic in nature. We hypothesize that idiosyncratic DILI occurs due to
crosstalk between drug reactive metabolite and cytokine stress signaling. To study
this hypothesis, human hepatoma HepG2 cells were exposed to diclofenac, which
causes idiosyncratic DILI in humans, in the presence of the pro-inflammatory cytokine
TNFα. Diclofenac itself induced a mild concentration-dependent apoptosis
of HepG2 cells. While TNFα itself was not cytotoxic, it strongly enhanced the diclofenac-induced
apoptosis. Using a siRNA screening approach and a live cell imaging
of apoptosis technique, diclofenac/TNFα-induced apoptosis was identified
as death-receptor dependent involving the intrinsic, mitochondrial death pathway.
In addition, diclofenac itself caused sustained activation of the stress kinase JNK,
and knock-down of this gene also resulted in an inhibition of the induced apoptosis.
Under normal conditions these two pro-apoptotic signaling pathways that are
activated down-stream of the TNF-receptor are controlled by the activation of the
transcription factor NF-κB and the resulting gene transcription. By using immunofluorescence
staining of wild type HepG2 cells and live cell imaging of
HepG2 cells expressing GFP-p65 we show that diclofenac causes a delay in the NFκB
oscillatory nuclear-to-cytosol translocation pattern in association with reduced
NF-κB transcriptional activity. The anti-apoptotic role of p65 was evident since
both inhibition of IKK as well as stable lentiviral shRNA-based knock down of p65
sensitized hepatocytes towards diclofenac/TNFα-induced cytotoxicity. Together
our data suggest a model whereby diclofenac-mediated stress signalling suppresses
SOT 2011 ANNUAL MEETING 87