The Toxicologist - Society of Toxicology

histone deacetylase that is necessary for peroxisome proliferator activated-receptor
alpha mediated gene induction. The purpose of the current study was to determine
whether tBHQ induces drug transporter expression in hepatocytes via a Sirt1-dependent
mechanism. Primary hepatocytes were isolated from male wild-type or hepatocyte-specific
Sirt1 knockout mice (Sirt1-KO) by a two-step perfusion. After
plating on collagen-coated plates, cells were treated with tBHQ (100μM) for 24
hours, and then total RNA was isolated. The mRNA expression levels were quantified
by quantitative real-time PCR. tBHQ increased Peroxisome proliferator-activated
receptor alpha (Ppar-α),Peroxisome proliferator-activated receptor-gamma
coactivator-1α(Pgc-1α) as well as Nad(p)h:quinone oxidoreductase(Nqo1) and
multidrug resistance-associated protein (Mrp)2,3 mRNA expression in wild-type
hepatocytes, but this induction was attenuated in Sirt1-KO hepatocytes. In summary,
tBHQ is a known activator of the Nrf2 signaling pathway in primary hepatocytes.
This induction was attenuated in the Sirt1-KO hepatocytes, which may be
due to decreased Pgc1-α and Ppar-α expression or Sirt1-dependent modulation of
Nrf2 activity. (NIH 3R01ES016042)
415 RESPONSE OF NRF2-MODULATED GENES TO
CIGARETTE SMOKE.
B. Kosmider, E. Messier, H. Chu and R. J. Mason. National Jewish Health,
Denver, CO.
Oxidative stress caused by cigarette smoke (CS) directly causes lung injury and cell
death in chronic obstructive pulmonary disease. Moreover, many studies show that
second-hand smoke can have harmful effects on nonsmokers and even cause them
to develop lung cancer and heart disease. The epithelium is the barrier between inhaled
air, which contains toxic compounds in cigarette smoke and the underlying
tissue. To study CS-induced cell injury we analyzed human primary alveolar type II
(ATII) cells and alveolar macrophages isolated from deidentified healthy, nonsmoker,
smoker and ex-smoker lung donors. We compared expression of Nrf2, and
its repressor Keap1 and Nrf2-dependent genes such as HO-1 and NQO1 in these
cells by western blotting, RT-PCR and immunocytofluorescence. We found higher
Nrf2 expression in smokers and ex-smokers in comparison with non-smokers.
Moreover, we also analyzed Nrf2 localization in ATII cells and in lung tissue. We
observed Nrf2 translocation to the nucleus in smokers in comparison with nonsmokers.
This suggests Nrf2 activation by reactive oxygen species (ROS) induced
by cigarette smoke. These results indicate oxidative stress and DNA damage.
Identification of the pathways whereby cigarette smoke generates ROS and induces
apoptosis may further our understanding of the pathogenesis of cigarette smoke-induced
lung disease. This work is supported by a Young Clinical Scientist Faculty
Award to Beata Kosmider from the Flight Attendant Medical Research Institute.
416 INDIRUBIN-3’-(2, 3 DIHYDROXYPROPYL)-
OXIMETHER (E804) IS A POTENT AHR-AGONIST AND
MODULATOR OF INFLAMMATION PROFILES IN LPS-
TREATED RAW264.7 MACROPHAGES.
A. S. Babcock and C. D. Rice. Biological Sciences, Clemson University, Clemson, SC.
Multiple indirubins have been synthesized and shown to inhibit cyclin-dependent
kinases (CDKs) and glycogen-synthase kinase (GSK-3β) with varying degrees of
potency. Several indirubins are also aryl hybrocarbon receptor (AhR) agonists, and
as with CDK and GSK-3β inhibitory activities of indirubin derivatives, AhR activities
cover a wide range of potency. One indirubin in particular, Indirubin-3’-(2,3
dihydroxypropyl)-oximether (E804) demonstrates novel activity against STAT3
signaling. This is significant because STAT3 signaling is the primary means by
which IL-10 mediates its anti-inflammatory actions. Furthermore, IFN-γ induced
MHC-II expression and antigen presentation are suppressed by IL-10 through
STAT3 signaling. To date, there are no published studies describing the effects of
E804 on AhR signaling, nor has this compound been investigated for its potential
as an anti-inflammatory agent in macrophage-targeted initiatives to modulate inflammation.
We found that E804 induces the expression of CYP1A, AhR, and
COX-2 in the murine macrophage cell line RAW264.7 in a manner similar to that
induced by PCB-126. Induction of COX-2 by E804 suggests a pro-inflammatory
property, therefore the expression of iNOS, IL-6, TNF-alpha, and p65 in cells cotreated
with LPS were examined, but found to be suppressed. Using a focus qRT-
PCR array, we compared E804 and PCB-126 for their effects on expression of a
suite of genes associated with inflammation and toxicity. In most cases those genes
up-regulated by LPS treatment are suppressed by E804 and PCB-126. Collectively,
these data indicate that E804 is a potent AhR ligand and modulator of proinflammatory
profiles in the murine macrophage line RAW264.7 treated with LPS. As
with other potent AhR ligands, E804 may exert its effects by altering developmental
pathways: in macrophages this may contribute to differences between M1- and
M2- like phenotypes.
417 REFORMING THE TOXIC SUBSTANCES CONTROL
ACT (TSCA): CHALLENGES, OPPORTUNITIES, AND
TIMING.
T. Lewandowski 1 and S. Barone 2 . 1 Gradient, Seattle, WA and 2 U.S. EPA,
Washington, DC.
The Toxic Substances Control Act (TSCA) of 1976 significantly changed the regulatory
landscape for chemicals in the United States. It gave the federal government
greater powers to track the introduction of new chemicals into the consumer marketplace,
provided the U.S. EPA with limited powers to require testing of new
chemicals, and allowed for case-by-case restriction of chemicals shown to be particularly
hazardous. Yet the basic provisions of TSCA are now 35 years old, and unlike
most major environmental laws passed in the 1970s, the TSCA legal framework remains
essentially unchanged. Achieving the goals of TSCA, namely to ensure that
adequate data are available to allow assessment of the effect of chemical substances
and mixtures on health and the environment has also proven difficult. The regulatory
apparatus has been unable to cope with the large number of chemicals requiring
evaluation, has focused on new rather than existing chemicals, has largely ignored
the potential interaction of chemicals occurring in mixtures, and has
generally failed to keep up with advances in technology. While once a model regulation
for other nations, the TSCA framework has now been superseded by chemical
safety regulations adopted by other jurisdictions (e.g., REACH). Given the dramatic
changes in chemical technology, toxicology, and risk assessment that are
expected to occur in the near future, reform of TSCA is seen as a high priority by
many stakeholders. Issues that are likely to be addressed by reform of TSCA include
the distinct toxicology and exposure scenarios posed by nanotechnology, the possibility
of basing hazard identification for the large number of chemicals requiring assessment
on mechanistic and in vitro data rather than standard animal tests, and
consideration of special population groups (not only children but also those with
genetic susceptibilities or chronic health conditions). Reform of TSCA has the potential
to greatly affect the way chemical risks are assessed in the U.S. and may
therefore have a significant impact on the daily lives of society members.
418 THE INTERNATIONAL COOPERATION ON
ALTERNATIVE TEST METHODS (ICATM):
TRANSLATING SCIENCE TO PROVIDE IMPROVED
PUBLIC HEALTH SAFETY ASSESSMENT TOOLS.
W. Stokes 1 and M. Wind 2 . 1 NICEATM, NIEHS, Research Triangle Park, NC and
2 Consumer Product Safety Commission, Bethesda, MD.
In 2009, the United States, Canada, the European Union, and Japan signed a
Memorandum of Cooperation for International Cooperation on Alternative Test
Methods (ICATM). This agreement provides for enhanced international collaborations
for the validation, evaluation, and development of internationally harmonized
recommendations for new and alternative safety testing methods and strategies.
The initial participating validation organizations are the NICEATM and ICC-
VAM, ECVAM, JaCVAM, and Health Canada’s Environmental Health Science
and Research Bureau. A Korean Center for the Validation of Alternative Methods
(KoCVAM) has recently also been established and is in the process of becoming an
ICATM participant. The ICATM organizations work collaboratively to promote
the validation and regulatory acceptance of new, revised, and alternative test methods
that are based on sound science and that will provide continued or improved
protection of people, animals, and the environment while reducing, refining, and
replacing the use of animals where scientifically feasible. The participating validation
organizations seek to expand and strengthen cooperation, collaboration, and
communications on the scientific validation and evaluation of new alternative testing
methods proposed for regulatory health and safety assessments. Consistent collaborations
are critical to the design and conduct of validation studies, the evaluation
and independent scientific peer review of proposed test methods and the
development of harmonized recommendations for national and international regulatory
consideration. The enhanced international cooperation in these three areas is
expected to result in more efficient test method validation and review, and more
rapid national and international acceptance of scientifically valid test methods. We
will provide an overview of the ICATM process along with an introduction to each
participating validation organization and highlights of recent and planned ICATM
contributions.
SOT 2011 ANNUAL MEETING 89