The Toxicologist - Society of Toxicology

769 PARAQUAT-INDUCED CHANGES IN EARLY PHASE
PROTEIN EXPRESSION IN RAT LIVER
MITOCHONDRIA AND TISSUE HOMOGENATE.
P. Venkatakrishnan, E. S. Nakayasu, I. C. Almeida and R. T. Miller. Biological
Sciences, University of Texas at El Paso, El Paso, TX.
Paraquat (PQ) is a widely used synthetic herbicide and redox-cycling agent. Upon
one-electron reduction of PQ, the PQ . radical is formed. PQ radicals preferentially
accumulate in the lung via the action of polyamine transporters and cause pulmonary
edema. Case reports of PQ toxicity indicate that death is due to multisystem
organ failure, including hepatotoxicity. Earlier studies identified that PQ
causes reactive nitrogen species-mediated toxicity by affecting the complex 1 activity
of mitochondria and thus effects mitochondrial respiration at an early stage. The
aim of these studies was to identify the proteins that are modulated in the early
stage (3 hrs) following acute PQ exposure (40 mg/kg i.p). Proteomic analyses were
performed with purified mitochondria and liver tissue homogenate of PQ-treated
rats, using nanospray-coupled linear ion trap mass spectrometry in conjunction
with 18 O labeling. In mitochondria, MS analyses identified 29 and 8 proteins to be
down-, and up-regulated, respectively above 2 fold. In particular, peroxyredoxin 6,
galectin 9 and glutathione-s-transferase p were significantly down-regulated and
proteins such as mannosidase-2αB1 and thioredoxin-1 were up-regulated.
Immunochemical analyses confirmed that peroxyredoxin 6 levels were down-regulated
in mitochondria. GO ontology and Interpro analyses of these data indicated
that the proteins affected were mainly from the classes of oxidoreductase, transferase,
hydrolase and transport proteins. MS analyses of liver tissue homogenate
samples identified 20 and 17 proteins to be down- and up-regulated, respectively,
by more than 2 fold. Further studies are underway in order to understand the roles
of the modulated proteins in mitochondria and tissue homogenates during PQ toxicity
(Supported by ES011982 from NIEHS/NIH to RTM and 5G12RR008124
from NCRR to UTEP)
770 LYSOSOMAL IRON RELEASE ENHANCES CELL
KILLING AFTER PHOTODYNAMIC THERAPY
MEDIATED BY A MITOCHONDRIA-TARGETED
PHOTOSENSITIZER IN CANCER CELLS.
S. Saggu, G. Quiogue, H. Hung, J. J. Lemasters and A. Nieminen. Medical
University of South Carolina, Charleston, SC.
In photodynamic therapy (PDT), light activates a photosensitizing drug added to a
tissue, resulting in singlet oxygen formation and cell death. The photosensitizer, phthalocyanine
4 (Pc 4), localizes primarily to mitochondrial membranes in cancer
cells, resulting in mitochondria-mediated cell death. Another Pc 4 derivative, Pc
181, accumulates into lysosomes. In comparison to Pc 4, Pc 181 is a more effective
photosensitizer at promoting killing cancer cells after PDT. To assess further how
lysosomes contribute to PDT, we monitored cell killing of A431cells after PDT in
the presence and absence of bafilomycin, an inhibitor of the acidic vacuolar proton
pump that collapses the pH gradient of the lysosomal/endosomal compartment.
Bafilomycin by itself was not toxic but greatly enhanced Pc 4-PDT-induced cell
killing. In comparison, less enhancement of cell killing by bafilomycin occurred
after Pc 181-PDT at photosensitizer doses producing equivalent cell killing in the
absence of bafilomycin. To investigate whether iron loading of lysosomes affects
bafilomycin-induced cell killing, cells were incubated with ammonium ferric citrate
(0-30 μM) for 48 hours prior to PDT. Ammonium ferric citrate greatly enhanced
Pc 4 plus bafilomycin-induced cell killing without having toxicity by itself, indicating
that increasing the amount of chelatable iron stored in the lysosomes enhances
the efficacy of bafilomycin-mediated PDT. The iron chelators, desferal and starchdesferal,
and the inhibitor of mitochondrial calcium (and ferrous iron) uniporter,
Ru360, protected against Pc 4 plus bafilomycin toxicity. These results support the
hypothesis that lysosomal disruption can augment PDT with Pc 4, which targets
predominantly mitochondria, but less so after PDT with Pc 181, since Pc 181 already
targets lysosomes. Therefore, agents that disturb lysosomal function could
potentially be used as an adjuvant treatment with mitochondria-targeted photosensitizers.
Supported by CA119079.
771 CURCUMIN PROTECTS AGAINST ACRYLONITRILE-
INDUCED TOXICITY IN RAT ASTROCYTES VIA NF-E2
RELATED FACTOR 2 ACTIVATION.
X. Zhao 1 , R. Lu 1 and M. Aschner 2 . 1 Preventive Medicine, Jiangsu University,
Zhenjiang, Jiangsu, China and 2 Pediatrics/Toxicology, Vanderbilt University,
Nashville, TN.
Oxidative stress plays key role in acrylonitrile (AN) neurotoxicity. Accordingly, it is
important to address the efficacy of antioxidants in protecting against AN-induced
free radical damage. This study tested the hypothesis that pretreatment of cultured
neonatal rat cortical astrocytes with curcumin protects against oxidative damage
caused by AN, addressing potential mechanisms that are involved in activation of
the Nrf2 pathway and resultant induction of phase II detoxification enzymes as
well as antioxidant enzyme gene expression. Cortical astrocytes were pretreated
with 2, 5, 10, 20 uM Curcumin (CUR) 6 h prior to AN treatment (1 mM for 12
h). Pretreatments with CUR significantly increased cell viability and reduced cytotoxicity,
compared with astrocytes treated with AN alone. Analysis of markers of
oxidative stress and immunocytochemical detection of Nrf2 nuclear translocation
demonstrated that pretreatment with CUR decreased lipid peroxidation, increased
activities of SOD,CAT and cytochrome c oxidase, GSH contents and led to increased
Nrf2 expression and its nuclear translocation. Moreover, CUR also increased
expressions of HO-1 and gamma-GCS genes in the downstream of Nrf2.
Taken together, these studies establish that CUR stimulates Nrf2 activation and increases
antioxidant gene expression, thus offering a novel therapeutic modality to
attenuate oxidative stress produced by AN.
772 NRF-2 NULL MICE ARE MORE SUSCEPTIBLE TO 1-
BROMOPROPOANE-INDUCED HEPATOTOXICITY.
F. Liu 1 , S. Ichihara 2, 1 , S. Sheik Mohideen 1 , K. Itoh 3 , M. Yamamoto 4 , W. M.
Valentine 5 and G. Ichihara 1 . 1 Department of Occupational & Environmental
Health, Nagoya University Graduate School of Medicine, Nagoya, Japan, 2 Mie
University Graduate School of Regional Innovation Studies, Tsu, Mie, Japan,
3
Hirosaki University Graduate School of Medicine, Hirosaki, Aomori, Japan,
4
Tohoku University Graduate School of Medicine, Sendai, Japan and 5 Vanderbilt
University Medical Center, Nashville, TN.
Objective: 1-Bromopropane (1BP) was introduced as an alternative to ozone-depleting
solvents in the workplace. It was found that 1BP exhibits neurotoxicity, reproductive
toxicity and hepatotoxicity in rodents and recent occupationally intoxicated
cases revealed neurotoxicity of 1BP in humans. However, the mechanism
underlying the toxicities of 1BP has not yet been elucidated. The present study investigated
involvement of oxidative stress in 1BP hepatotoxicity using nuclear factor
erythroid 2-related factor 2(Nrf2) null mice. Methods: Each of 24 male mice of
Nrf2-null and wild type (WT) C57BL/6J were divided into three groups of eight
each and exposed to 1BP at 0, 100 and 300 ppm for 8 hr/day for 28 days by inhalation.
At the end of the exposure, the mice were decapitated and the liver was
dissected out immediately. A part of liver was fixed with neutral bufferized formalin
for histopathological studies and the remaining parts were frozen for biochemical
studies. Results: Liver histopathology showed a significantly larger area of liver
necrosis in Nrf2-null mice than WT mice. Nrf2-null mice showed higher malondialdehyde
(MDA) levels and higher ratios of GSSG/GSH as well as lower total glutathione
content, GPx activity and GST activity in the liver of Nrf2-null mice was
lower than WT mice. Exposure to 1BP at 300 ppm increased the mRNA expression
of HO-1, GST Yc2 and NQO1 in WT mice, but did not influence it in Nrf2-
null mice except GST Yc2. Conclusion: Nrf-2 null mice showed higher susceptibility
in the liver to 1BP exposure, being accompanied by higher oxidative stress in the
liver. The latter may be through lower expression of anti-oxidative bio-molecules in
Nrf-2 null mice. The results are consistent with the oxidative stress contributing to
1BP hepatotoxicity.
773 NRF2 PROTECTS AGAINST DIQUAT-INDUCED
TOXICITY.
K. C. Wu, Y. Zhang and C. D. Klaassen. University of Kansas Medical Center,
Kansas City, KS.
Diquat is a contact herbicide that generates superoxide anions through redox cycling.
Nrf2 is a transcription factor that up-regulates cytoprotective genes in response
to oxidative stress and electrophilic stimuli. To investigate the protective effect
of Nrf2 against diquat-induced toxicity, wild-type (WT), Nrf2-null, and
Keap1-knock down (Keap1-kd) mice (with enhanced Nrf2 activity) were treated
with diquat (125 mg/kg, i.p.). Blood and tissues were collected 1, 2, 4, and 6h
thereafter. At 6h, 30% of Nrf2-null mice, 50% of WT mice, and 100% of Keap1-
kd mice survived. Diquat caused lipid oxidation in WT mice, as evidenced by elevated
serum thiobarbituric acid-reactive substances (TBARS), which was higher
Nrf2-null, but lower in Keap1-kd mice. Diquat-induced hepatotoxicity was indicated
by increased serum ALT activity in all three genotypes, with Nrf2-null having
higher and Keap1-kd having lower serum ALT than WT mice. Histological examination
after diquat treatment indicated extensive necrosis in livers of Nrf2-null
mice, but no morphological changes in livers of WT or Keap1-kd mice. Six hours
after diquat treatment, Nrf2-null mice had more severe lung toxicity than WT
mice, as evidenced by increased lung weight, as well as more alveolar collapse. In
contrast, Keap1-kd mice had attenuated lung edema and no apparent histopathology.
To further investigate the mechanism of the protective effect of Nrf2, lung and
liver glutathione (GSH) and GSSG concentrations were quantified by UPLC-
MS/MS. Nrf2-null had lower and Keap1-kd had higher levels of GSH than WT
mice before treatment. Diquat treatment decreased GSH in lung and liver in WT
164 SOT 2010 ANNUAL MEETING