The Toxicologist - Society of Toxicology

1293 TRANSFORMING GROWTH FACTOR BETA MEDIATES
OZONE-INDUCED AIRWAY WALL FIBROSIS.
R. Liu 1 , A. Katre 1 , C. Ballinger 1 , H. Akhter 1 , M. Fanucchi 1 , D. Kim 2 and E.
Postlethwait 1 . 1 University of Alabama at Birmingham, Birmingham, AL and 2 Ewha
Womans University, Seoul, Republic of Korea.
Background: Ozone (O3), a commonly encountered environmental pollutant, has
been shown to induce pulmonary fibrosis in different animal models; the underlying
molecular mechanism, however, remains elusive. Methods: Six to eight week
old male C57BL mice were exposed to a cyclic O3 exposure protocol consisting of
2 days of filtered air and 5 days of O3 exposure (0.5 ppm, 8 hrs/day) for 5 and 10
cycles with or without intra-peritoneal injection of IN-1233, an inhibitor of type 1
receptor of transforming growth factor beta (TGF-β), the most potent profibrogenic
cytokine. Results: The results showed that O3 exposure for 5 or 10 cycles increased
the TGF-β protein level in the bronchoalveolar lavage fluid (BALF), associated
with an increase in the expression of plasminogen activator inhibitor 1 (PAI-1),
a TGF-β responsive gene which plays a critical role in the development of fibrosis.
Cyclic O3 exposure also increased the deposition of collagens and alpha smooth
muscle actin (alpha-SMA) within airway walls. However, these fibrotic changes
were not overt until after 10 cycles of exposure. We further showed that treatment
of mice with IN-1233 suppressed O3-induced PAI-1 expression and fibrotic
changes. Conclusion: Our data demonstrate for the first time that activation of the
TGF-β signaling pathways mediates O3-induced lung fibrotic responses in vivo.
1294 ANGIOTENSIN II PLAYS A CRITICAL ROLE IN
DIABETIC LUNG FIBROSIS VIA ACTIVATION OF
NADPH OXIDASE-MEDIATED NITROSATIVE
DAMAGE.
L. Cai 1, 2 , J. Yang 2, 1 , Y. Tan 1 and L. Miao 2 . 1 Pediatrics, University of Louisville,
Louisville, KY and 2 Second Hospital of Jilin University, Changchun, China.
Diabetic patients have a high risk of pulmonary disorders that are usually associated
with restrictive impairment of lung function, suggesting a fibrotic process. Human
pathological examination on the hung from diabetic patients supports the existence
of lung fibrosis. However, it is remains unclear whether diabetes is able, and how to
induce lung fibrosis. The present study was undertaken to define whether and how
diabetes causes lung fibrosis. Lung samples from streptozotocin-induced type 1 diabetic
mice, spontaneously developed type 1 diabetic OVE26 mice, and their agematched
controls, were investigated with histopathological and biochemical analysis.
Signaling mechanisms were also explored with cultured normal human lung
fibroblasts in vitro. In diabetes, histological examination with Sirius-red staining
and H & E staining showed significant fibrosis, along with massive inflammatory
cell infiltration. Lung fibrosis and inflammation were confirmed by real time PCR
and Western blotting assays for the increased fibronectin, CTGF, PAI-1 and TNFα
mRNA and protein expressions. Diabetes also significantly increased NADPH
oxidase (NOX) expression and protein nitration, along with up-regulation of angiotensin
II (Ang II) and its receptor expression. In cell culture, exposure of lung fibroblasts
to Ang II increased CTGF expression in a dose- and time-dependent
manner, which could be abolished by inhibition of superoxide, NO, and peroxynitrite
accumulation. Furthermore, chronic infusion of Ang II to normal mice at a
subpressor dose induced diabetes-like lung fibrosis, and Ang II receptor AT1
blocker (losartan) abolished the lung fibrotic and inflammatory responses in diabetic
mice. These results suggest that Ang II plays a critical role in diabetic lung fibrosis,
which is caused by NOX activation-mediated nitrosative damage.
1295 THE ROLE OF NITRIC OXIDE SYNTHASE IN
PARAQUAT-INDUCED OXIDATIVE STRESS IN A
SKELETAL MUSCLE CELL LINE.
P. Venkatakrishnan, S. Panda, L. Roman and B. S. Masters. Biochemistry,
University of Texas Health Science Center, San Antonio, TX.
Nitric Oxide Synthase (NOS) is a heme-containing flavoenzyme that catalyzes the
conversion of L-arginine to L-citrulline, and NO . is formed as the co-product. The
function of NO . varies depending on the NOS isoform and the location at which it
is being produced. NO . produced by nNOS facilitates neurosignaling, by eNOS,
vasodilation, and iNOS, cytoprotection from foreign agents. nNOSμ, a splice variant
form of nNOS containing an extra 34 amino acid insert between exon 16 and
17, was identified to be present in the skeletal muscle and heart. In skeletal muscle,
nNOS is complexed with the Dystrophin-Glycoprotein Complex (DGC) where it
is bound through α-syntrophin. Although the function of this NOS isoform in the
skeletal muscle is poorly understood, nNOS membrane localization with DGC is
critical for vasoconstriction and in case of Duchenne Muscular Dystrophy, nNOS
is found to be displaced from the sarcolemma. In order to understand the role of
nNOSμ in the regulation of antioxidants in skeletal muscle during aging, we exam-
ined the functional significance of nNOSμ in C2C12 myoblast cell line under conditions
of oxidative stress using paraquat(PQ). C2C12 cells were subjected to
serum starvation and thus allowed to differentiate to form myotubes. Myotubes
were pretreated with L-NAME(3 mM), an nNOS inhibitor, for 45 mins and later
with PQ(100 μM) for various time points (2, 4, 8, 12, 24 and 48, 72 hrs) with appropriate
controls. Morphological changes of myotubes clearly indicate that L-
NAME has a protective effect on PQ-induced damage at intermediate time points.
Cell lysates were collected at these time points for Western Blotting to probe for βdystroglycan,
nNOSα (also known as nNOS), nNOSμ and oxidative stress markers
such as peroxyredoxin 6. In addition, the similarity and differences in the enzymatic
activity in terms of electron transfer efficiency between nNOSα and nNOSμ
were studied.
1296 GENERATION OF ACETYL RADICAL AND SINGLET
OXYGEN BY REACTIVE ALPHA-OXOALDEHYDES
LINKED TO AGING AND DIABETES.
J. Massari 1 and E. J. Bechara 1, 2 . 1 Ciências Exatas e da Terra, Universidade Federal
de São Paulo, Diadema, São Paulo, Brazil and 2 Biochemistry, São Paulo University,
São Paulo, São Paulo, Brazil. Sponsor: S. Berlanga Barros.
Diacetyl (DA) - a food contaminant, methylglyoxal (MG) - an endogenous toxin in
diabetes, and glyoxal (GO) – a metabolite implicated in ageing, are increasingly
more reactive than monocarbonyls regarding nucleophilic additions to proteins and
DNA. We have recently reported that DA e MG undergo attack by peroxynitrite, a
strong biological oxidant and nucleophile, in phosphate buffer pH 7, followed by
oxidation by oxygen. The reaction putatively involves the intermediacy of a nitrosoperoxo
adduct, followed by homolysis to an oxyl radical, β-scission to acetyl
radical, and ultimately formation of acetate (from DA) or acetate plus formate
(from MG. These reactions could be coupled to radical acetylation of added amino
acids and nucleobases, leading us to hypothesize that they may be involved in posttranslational
protein modifications and mutagenesis. We now show that at identical
experimental conditions GO reacts with peroxynitrite to yield mainly formate at
rates at least one order of magnitude higher than MG. The novelty here, predicted
by assuming subsequent formation of a formyl radical from GO, dioxygen insertion,
and Russell mechanism reaction, is the detection of the singlet molecular oxygen
monomol light emission at 1270 nm during the reaction. Accordingly, the
infra-red emission area increases upon raising the concentration of peroxynitrite, is
suppressed upon addition of singlet quenchers (azide, His), is enhanced in deuterated
buffer, and is not observed with DA, a source of acetyl, but not formyl radical.
Chemical trapping of singlet oxygen using anthracene-9,10-divinylsulfonate was
also performed. Our studies may add insights to detail the molecular events underlying
nitrosative, oxidative and carbonyl unbalance in some disorders. Support:
FAPESP, CNPq, INCT Redoxoma.
1297 CARBONYLATION AND INACTIVATION OF
MITOCHONDRIAL COMPLEX I DURING KAINATE-
INDUCED EPILEPTOGENESIS.
K. Ryan and M. Patel. Pharmaceutical Sciences, University of Colorado Denver,
Aurora, CO.
Nearly 1% of the population suffers from epilepsy. Acquired epilepsies such as temporal
lobe epilepsy (TLE) resulting from trauma, chemical exposure or infections
comprise a majority of epilepsy cases. Like many progressive disorders, mitochondrial
dysfunction and oxidative stress are linked to the development of TLE. A single
injection of kainate, an excitotoxic agent, results in acute excitotoxicity and
chronic development of spontaneous seizures via a process termed epileptogenesis.
We have previously shown increased mitochondrial-specific reactive oxygen species
(ROS) production, mitochondrial DNA damage and decreases in mitochondrial
redox status in the rat hippocampus in the kainate model of epileptogenesis (Jarrett
S et al 2008; Liang L et al 2006). We hypothesize that Complex I (CI) dysfunction
may be the primary cause of mitochondrial inhibition and contribute to seizures:
increasing evidence suggests that ROS-induced protein post-translational modifications
are a contributing factor. The goal of this study was to determine if the mechanism
of CI dysfunction is related to oxidative modification of the 75kDa subunit
(Ndusf1) within the complex. Rats were injected with kainate or vehicle and monitored
by video and EEG for seizure activity for 6 weeks. Mitochondrial CI activity
was decreased 2 d and 6 weeks after kainate injection; whereas ATP levels were persistently
decreased throughout the 6 week period. Evidence of CI modifications was
measured throughout epileptogenesis. CI was immunoprecipiated from hippcomapal
tissue and subunits were separated by SDS-PAGE. Ndusf1 modifications were
identified with an Agilent 6510 QTOF LC/MS system and MS/MS data was analyzed
with Mascot 2.2 (Matrix Science). In this study, we observed a time-dependent
increase in Ndusf1 carbonylation throughout the course of epileptogenesis.
This irreversible modification has been considered a biomarker for oxidative-induced
cellular damage and may be a future therapeutic target in the prevention of
epileptogenesis.
SOT 2011 ANNUAL MEETING 277