The Toxicologist - Society of Toxicology

261 NITRIC OXIDE ENHANCES BACTERIAL CLEARANCE
IN HYPEROXIA.
A. Gore. St. John’s University, Queens, NY.
A significant number of patients on ventilator develop ventilator-associated pneumonia
(VAP) and inflammatory lung injury. Despite the advancement of antimicrobial
therapy, VAP results in significant morbidity and mortality in these patients.
Previously, studies including ours have demonstrated exposure to prolonged hyperoxia
results in macrophage dysfunction. In this study, we observed that prolonged
hyperoxia suppressed levels of airway NO. We tested whether administration of exogenous
NO enhances the host’s ability to clear the bacteria. C57BL/6 mice were
randomized to receive either intranasal DETA-NONOate (D-NO), a NO donor,
or control solution, exposed to ≥99.0% O2, and then inoculated with intranasal instillation
of PA. Treatment with D-NO improved the survival, decreased bacterial
load in the lung and mitigated hyperoxia/PA-induced lung injury in a dose-dependent
manner. Moderate concentrations of D-NO significantly rescued hyperoxia-suppressed
macrophage functions in RAW 264.7 cells exposed to 95% O2.
However, macrophages exposed to D-NO at concentrations of 150 μM or higher
underwent cell death. Our previous work demonstrates that hyperoxia induces
HMGB1 release, which contributes to hyperoxia-compromised macrophage dysfunction
and is partly mediated by NF-κB. D-NO inhibited hyperoxia-induced
HMGB1release and NF-κB activation in RAW 264.7 cells. These results indicate
that exogenous NO might provide a novel therapeutic approach in preventing patients
on ventilator to develop VAP.
262 ENDOTHELIAL ACTIVATION BY 4, HYDROXY TRANS-
2-NONENAL: ROLE OF ENDOPLASMIC RETICULUM
STRESS.
E. N. Vladykovskaya, S. D. Sithu, P. Haberzettl, N. S. Wickramasinghe, O. A.
Barski, S. E. D’Souza, A. Bhatnagar and S. Srivastava. School of Medicine,
University of Louisville, Louisville, KY.
Endothelial activation is an early event in atherogenesis. Although the mechanisms
leading to endothelial activation in atherogenesis remain poorly understood, extensive
evidence suggests that activation of the endothelium in lesion-prone sites is due
to the sub-endothelial activation of oxidized LDL. We observed that 4-hydroxytrans-2-nonenal
(HNE; 25 μM), one of the most abundant and reactive aldehyde,
generated from the oxidation of LDL and membrane lipids increased the cell surface
expression of ICAM-1 on endothelial cells (EC) by 1.3-fold, augmented
monocyte adhesion to EC (2-fold) and their transmigration through EC (2-fold),
and increased the expression of pro-inflammatory cytokines (TNF-α, IL-6 and IL-
8) by 4-12-fold. HNE also modified several proteins in EC. Confocal imaging of
HNE-treated EC showed that the modified proteins were associated with the ER.
Exposure to HNE led to the splicing of the bZIP transcription factor, XBP-1, a
characteristic feature of the alarm phase of ER stress, and an increase in the phosphorylation
of eIF2α, which regulates the expression of multiple ER-stress dependent
transcription factors. These transcription factors, ATF3 and ATF4, were also
strongly (3-5 fold) induced by HNE. Treatment with HNE was associated with a 2-
5 fold increase in the expression of ER chaperones, GRP78 and HERP, indicating
that HNE triggers the adaptive phase of the response, which increases the protein
folding capacity of the ER. Adenoviral transfection with ATF6 increased the expression
of molecular chaperones Grp78, Grp94 and PDI and attenuated HNE-induced
monocyte adhesion to EC and transcription of IL-8. Similarly, pre-treatment
with the chemical chaperone, phenylbutyric acid (PBA), to facilitate protein folding,
attenuated HNE- induced monocyte adhesion and transcription of IL-8.
These data suggest that HNE- induced ER stress could be a critical regulator of endothelial
function.
263 LOVASTATIN ATTENUATES HYPEROXIA-INDUCED
HMGB1 RELEASE AND RESCUES MACROPHAGE
FUNCTION VIA INACTIVATION OF NF-κB P65
PATHWAY.
R. Malla 1 and L. Mantell 1, 2 . 1 St. John’s University, Queens, NY and 2 The Feinstein
Institute for Medical Research, North Shore-LIJ Health System, Manhasset, NY.
Mechanical ventilation with supraphysiological concentrations of oxygen (hyperoxia)
is routinely used in treating both newborns and adults with respiratory insufficiency.
However, exposure to prolonged hyperoxia is associated with increased
susceptibility to microbial infection. Our previous studies also demonstrate that
HMGB1 (a pro-inflammatory cytokine) from macrophages exposed to hyperoxia
results in decreased ability to clear micro-organisms in mouse model of
Pseudomonas aeruginosa. Statins, HMGcoA reductase inhibitors, have been shown
to reduce elevated inflammatory cytokines like IL-6, IL-8 and TNF-alpha in hypercholesterolemic
patients. The purpose of this study was to investigate whether
56 SOT 2011 ANNUAL MEETING
Lovastatin, a Statin analogue, affects hyperoxia-induced release of HMGB1 and
function in cultured RAW 264.7 macrophages. We tested the hyperoxia-suppressed
ability of macrophages to phagocytose microbes by pretreatment with a series of
concentrations of Lovastatin and then expose to 95%O2 for 24 hr. Phagocytosis
and HMGB1 release was determined by immunofluorescence and Western Blot
analysis respectively. The release of HMGB1 under hyperoxic conditions was inhibited
by Lovastatin in a dose-dependent manner. Corresponding to the decreased
release of HMGB1, Lovastatin significantly enhanced macrophage’s ability to
phagocytose under hyperoxic conditions. To further investigate whether attenuating
effects of Lovastatin was NF-κB mediated, we studied NF-κB translocation on
hyperoxia-suppressed macrophages via immunofluorescence . These results indicate
that Lovastatin can effectively reduce both hyperoxia-induced inflammation mediated
by HMGB1 and rescue hyperoxia-suppressed macrophage function. Also, it
implicates that HMGB1 release is atleast partly mediated by NF-κB under hyperoxia.
Therefore, lovastatin might be clinically employed to treat patients on ventilation
with inflammatory lung injury and ventillator-associated pneumonia.
264 SELECTIVE ALPHA7 NICOTINIC ACETYLCHOLINE
RECEPTOR AGONIST GTS-21 ENHANCES HYPEROXIC
MACROPHAGE FUNCTION.
R. A. Sitapara. Department of Pharmaceutical Sciences, St. John’s University, Jamaica,
NY. Sponsor: L. Mantell.
Macrophages are the first line of defense against invading pathogens. However, exposure
to prolonged hyperoxia compromises host ability to phagocytose bacteria.
Our previous studies show that exposure to prolonged hyperoxia leads to HMGB1
release and HMGB1 suppresses macrophage’s ability to phagocytose. The vagus
nerve of autonomous nervous system regulates the levels of proinflammatory cytokines,
thus modulating inflammation-mediated damage. Dr. Kevin Tracey’s lab
has shown that 3(2,4 dimethoxybenzylidene) anabaseine (GTS-21), an α7 nicotine
acetylcholine receptor agonist inhibits endotoxin-induced HMGB1 release from
RAW 264.7 cells (a macrophage like cell line). The aim of this study was to examine
whether GTS-21 could inhibit hyperoxia-induced HMGB1 release and enhance
macrophage function under hyperoxic condition. RAW 264.7 cells were exposed
to different concentrations of GTS-21 prior to exposure to 95%
O 2 .Phagocytosis assay was performed to determine macrophage function. HMGB1
release was assessed by Western blotting and immunofluroscence analyses. Nuclear
factor kappa B (NFκB)has been shown to modulate endotoxin-induced HMGB1
release. Activation of NFκB was assessed by its translocation from cytoplasm to nuclei
by immunofluroscence assay. We show here that GTS-21 dose dependently inhibited
HMGB1 release and NFκB translocation, and enhanced the ability of
macrophage to phagocytose. These results indicate that GTS-21 is effective in inhibiting
hyperoxia-induced HMGB1 release and enhancing hyperoxic macrophage
function.
265 METALLOTHIONEIN AND ACUTE CADMIUM-
INDUCED DNA OXIDATIVE DAMAGE.
W. Qu 1 , L. Cheng 1 , Y. Sun 1 , R. P. Mason 2 and M. Waalkes 1 . 1 NTP Laboratories,
NTP, NIEHS, Research Triangle Park, NC and 2 Laboratory of Toxicology and
Pharmacology, NIEHS, Research Triangle Park, NC.
Metallothionein (MT) plays an important role in detoxication of heavy metals such
as cadmium (Cd), a human carcinogen. The mechanisms by which MT may impact
Cd carcinogenesis remain poorly understood. Thus, we studied the role of MT
in acute Cd-induced oxidative DNA damage (ODD). MT-I/II double knockout
(MT-null) and parental wild-type (WT) cell lines were treated with Cd (as CdCl2)
for 24 h and cytotoxicity was measured. Cd was much less cytotoxic in WT cells
(LC50 = 16.5 ± 2.1 μM, mean ± SEM) than MT-null cells (LC50 = 6.6 ± 1.6 μM).
Cd-induced ODD was measured using the immuno-spin trapping method which
directly traps DNA radicals by using a spin-trap agent to form a stable DNA nitrone
adduct in situ from the radicals. Cd treatment (1 or 5 μM; 24 h) induced
much less ODD in WT cells (114% and 137% of control, respectively) compared
to MT-null cells (298% and 438%). Cd caused concentration-related increases in
MT expression in WT cells (transcript and protein). In contrast, the basal levels of
MT were very low and were not increased by Cd exposure in MT-null cells. Cd
treatment increased expression of two important transport genes, Mrp1 and Mrp2,
in WT cells but not MT-null cells. Cd caused concentration-related increases in oxidant
defense genes, such as HO-1 and GSTa2, in both WT and MT-null cells.
However, the increased expression was much higher in MT-null cells, where, for example,
both HO-1 and GSTa2 maximal transcript levels after Cd treatment were 3fold
higher than WT cells. Treatment with the direct oxidant, hydrogen peroxide,
at 0.25 and 0.5 mM (24 h) also caused a concentration-dependent increase of