The Toxicologist - Society of Toxicology

mice; the decrease was more pronounced in Nrf2-null and less in Keap1-kd mice.
After diquat treatment, the mRNA of the GSH synthesis enzyme Gclc was increased
in Keap1-kd, but not in Nrf2-null mice. In conclusion, Nrf2 activation
lowers lipid peroxidation products in the circulation and prevents liver and lung injury
from diquat, likely due to higher concentrations of GSH, and induction of
genes involved in detoxication. (Supported by NIH grants ES009649, ES013714,
ES009716, ES007079, and RR021940)
774 ACTIVATION OF YAP1 BY HYDROGEN PEROXIDE OR
CYSTEINE THIOL-REACTIVE MICHAEL ACCEPTORS
LEADS TO SPECIFIC ADAPTIVE GENE RESPONSES IN
THE YEAST SACCHAROMYCES CEREVISIAE.
X. Ouyang, N. Q. Tran, C. Sutter and T. R. Sutter. W. Harry Feinstone Center for
Genomic Research, University of Memphis, Memphis, TN.
The yeast Saccharomyces cerevisiae transcription factor Yap1 mediates an adaptive
response to oxidative stress by inducing the expression of a large number of antioxidant
genes. When yeast cells are pretreated with a sublethal dose of an oxidant,
Yap1 translocates from the cytosol into the nucleus and confers the cells with a resistance
against a later challenge from an oxidant at higher concentrations. There
are two known mechanisms by which Yap1 may be activated. H2O2 activates Yap1
though a Gpx3-dependent pathway, involving the formation of disulfide bonds between
cysteines in the N-terminal and C-terminal cysteine rich domains (CRDs) of
Yap1, whereas the thiol-reactive N-ethylmaleimide (NEM) activates Yap1 via a
Gpx3-independent mechanism that requires only the C-CRD cysteines of Yap1. In
this study, we demonstrate that H2O2 and NEM show no cross protection to each
other. Another thiol-reactive chemical, acrolein, also induces a Yap1-mediated
adaptive response and can cross protect against NEM, but not H2O2. Cellular localization
and functional experiments using yeast strains with mutant YAP1 genes
fused to the green fluorescence protein gene indicate that NEM and acrolein activate
Yap1 through the same mechanism requiring two of the C-CRD cysteines. By
microarray analysis we identify two sets of Yap1-dependent genes that respond
specifically to H2O2 or to NEM and acrolein. By functional analysis using yeast
single deletion strains we identify genes in each set that provide resistance against
the corresponding inducers. These data demonstrate that Yap1 is sensitive to different
types of oxidative stress through distinct CRD-mediated mechanisms and responds
accordingly with selective expression of protective genes.
776 CALIFORNIA WILDFIRES OF 2008: LUNG
ANTIOXIDANT RESPONSE TO COARSE AND FINE
PARTICULATE MATTER.
T. C. Wegesser 1 , L. M. Franzi 1 , M. M. Frank 2 , E. Arantza 3 and L. A. Jerold 1 .
1
Pulmonary and Critical Care Medicine, University of California Davis, Davis, CA,
2
Animal Science, University of California Davis, Davis, CA and 3 COEH-SCPC
Department, University of California Los Angeles, Los Angeles, CA.
In 2008 California experienced a major outbreak of wildfires with transport of
smoke over large distances, especially in the Central Valley. Coarse (PM2.5-10) and
fine (PM2.5) particulate matter (PM) concentrations were greatly in excess of the
air quality standards. We previously reported that wildfire-derived coarse or fine
PM intratracheally instilled into lungs of mice induce a strong inflammatory response
(Wegesser et al., 2009, Environmental Health Perspectives 117:893-897).
Pro-inflammatory activity of PM and measured levels of antioxidant capacity in recovered
lavage fluid are correlated. Coarse PM is inflammatory at doses as low as 10
ug/mouse, with a NOAEL at 1 ug/mouse. Neutrophil chemokines/cytokines and
TNF-α were elevated in the lung lavage fluid obtained 6 and 24 hours after PM instillation,
consistent with the neutrophilic inflammatory response observed 24
hours after PM administration. Chemical analysis of the PM preparations resulted
in relatively low polycyclic aromatic hydrocarbons (PAHs) content as compared to
published results from typical urban PM. The coarse PM fraction is more active on
an equal dose basis than the fine PM despite its lower content of PAHs. We did not
find any correlation between the content of any specific PAH (or total PAH content)
in the PM fraction and PM toxicity. Concentrations of the oxidation products
of phenathrene and anthracene, phenathraquinone and anthraquinone, were several-fold
higher in the coarse than the fine fraction, suggesting a significant role for
atmospheric photochemistry in the formation of secondary pollutants in the wildfire
PM and the possibility that such secondary pollutants could be important
sources of toxicity in the wildfire PM. We now demonstrate that wildfire PM also
causes major increases in oxidative stress in mouse lungs as measured by decreased
antioxidant content in lung lavage fluid.
777 UP-REGULATION OF HEME OXYGENASE-1 IN RAT
SPLEEN FOLLOWING ANILINE EXPOSURE.
J. Wang 1 , H. Ma 1 , P. J. Boor 1 , V. M. Sadagopa Ramanujam 2 , G. A. Ansari 1 and
M. Khan 1 . 1 Pathology, University of Texas Medical Branch, Galveston, TX and
2
Preventive Medicine and Community Health, University of Texas Medical Branch,
Galveston, TX.
Aniline exposure causes toxicity to the spleen which is characterized by vascular
congestion, hyperplasia, fibrosis and development of a variety of sarcomas in rats.
However, underlying mechanisms by which aniline elicits splenotoxic response are
not well understood. Previously we have shown that aniline exposure causes oxidative
damage to the spleen. To further explore the oxidative mechanism of aniline
toxicity, we evaluated the potential contribution of heme oxygenase-1 (HO-1),
which catalyzes heme degradation and releases free iron. Male SD rats were given 1
mmol/kg/day aniline in water by gavage for 1, 4 or 7 days, while respective controls
received water only. Aniline exposure led to significant increases in HO-1 mRNA
expression in the spleen (2- and 2.4-fold at days 4 and 7, respectively) with corresponding
increases in protein expression, as confirmed by ELISA and Western blot
analyses. Furthermore, immunohistochemical assessment of spleen showed
stronger immunostaining for HO-1 in the spleens of rats treated for 7 days, confined
mainly to the red pulp areas. No changes were observed in mRNA and protein
levels of HO-1 following 1 day exposure. The increase in HO-1 expression was
associated with increases in total iron (2.4- and 2.7- fold), free iron (1.9- and 3.5-
fold), and ferritin levels (1.9- and 2.1-fold) at 4 and 7 days of aniline exposure. Our
data suggest that HO-1 up-regulation in aniline-induced splenic toxicity could be a
contributing pro-oxidant mechanism, mediated through iron release, and leading
to oxidative damage. Supported by ES06476.
778 RADICAL MECHANISMS IN NITROSAMINE AND
NITROSAMIDE-INDUCED GENE EXPRESSION
MODULATIONS IN CACO-2 CELLS.
D. Hebels, J. J. Briedé, R. Khampang, J. C. Kleinjans and T. M. de Kok. Health
Risk Analysis and Toxicology, Maastricht University, Maastricht, Netherlands.
Sponsor: H. van Loveren.
Nitrosamines and nitrosamides, two classes of N-nitroso compounds (NOC), may
be implicated in human colon carcinogenesis following gastro-intestinal nitrosation
processes. Since genotoxic concentrations of nitrosamines and nitrosamides were
previously found to result in distinct effects on gene expression modulation in
colon cells in vitro, in particular pathways involved in oxidative stress, we hypothesized
that differences in radical generation are responsible for these discriminative
transcriptomic responses. To investigate the radical generating capacity of NOC in
a cellular system, the human colon adenocarcinoma cell line Caco-2 was exposed to
genotoxic concentrations of the nitrosamides N-methyl-N’-nitro-N-nitrosoguanidine
(MNNG, 1 μM) and N-methyl-N-nitrosurea (MNU, 1 mM), and the nitrosamines,
N-nitrosodiethylamine (NDEA, 50mM), N-nitrosodimethylamine
(NDMA, 100 mM), N-nitrosopiperidine (NPIP, 40 mM), and N-nitrosopyrrolidine
(NPYR, 100mM) for 30 minutes and measured by ESR spectroscopy.
Nitrosamine exposure resulted in the formation of reactive oxygen species (ROS)
and a carbon centered radical, identified as the α-nitrosamino radical, which was
catalyzed by the presence of cells, thus suggesting the need for metabolic activity.
MNU exposure resulted in a small ROS signal, and formation of a nitrogen centered
radical (NCR), the amidyl radical, also catalyzed by presence of cells. MNNG
did not influence radical formation, but at a concentration of 1mM, exposure resulted
in the formation of both ROS, as well as, in NCR formation. Nitrosamines
no longer displayed any radical formation at this concentration. By associating gene
expression patterns with ROS formation, we identified several cellular processes including
apoptosis, cell cycle blockage, DNA repair and oxidative stress. Cellular
processes were only affected by nitrosamines, analogous to the difference in ROS
levels, suggesting that ROS formation plays an important role in the gene expression
effects following NOC exposure in Caco-2 cells.
779 POST-TRANSLATIONAL MODIFICATION AND
REGULATION OF GLUTAMATE CYSTEINE LIGASE BY
THE α, β-UNSATURATED ALDEHYDE 4-HYDROXY-2-
NONENAL (4-HNE).
D. Backos 1 , K. S. Fritz 1 , J. R. Roede 2 , D. R. Petersen 1 and C. C. Franklin 1 .
1
Toxicology, University of Colorado Denver, Aurora, CO and 2 Pulmonology, Emory
University School of Medicine, Atlanta, GA.
Reactive α,β-unsaturated aldehydes generated from the oxidation of polyunsaturated
fatty acids are characteristic of many human diseases associated with oxidative
stress. 4-hydroxy-2-nonenal (4-HNE) is a major α,β-unsaturated aldehyde produced
during lipid peroxidation and has the ability to modify protein function via
formation of covalent adducts on nucleophilic amino acid residues with a relative
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