Proantosiyanidinin nitrojen mustarda maruz bırakılmış künt travma oluşturulmuş akciğerlerdeki etkisi (Gerçek terör saldırı simülasyonu)

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Introduction
One of the most important chemical warfare agents
is nitrogen mustard (NM) (1,2). It is also known as
NM, and it is the most widely used agent in chemical weapons (3,4). Sulfur mustard (SM) has posed a
military threat and this is still considered as a major threat agent for mankind (5,6). NM is a structural
analogue of SM (1). Many agreements were spurred
after the Second World War for the prohibition of
the manufacture and use of NM. These are the 1993
Chemical Weapons Convention (CWC) and chemical warfare agent (CWA) destruction programs (7).
Currently, instead of studying CWA many investigators have preferred dealing with their analogs due to
the following reasons: 1- the toxicity of some analogs
has been recognized to be similar or even greater than
that of the parent CWA, and 2- the increased concern
that CWA could be used by terrorists against civilians (8,9). In addition, chemical agents are used to
amplify the injurious power of the destroying weapons. As chemical weapon-mediated terrorist attacks
are common nowadays, experimental studies should
be carried out for CWA-s.
The most destructive effects of mustards occur on
the respiratory system, eyes and skin (5,7,10). Because
of their significant effects on respiratory system, mustards are extremely relevant to trauma-dealing medical staff too. NM exposure and blunt thorax trauma
(BTT) also cause inflammatory lung diseases, including acute respiratory distress syndrome (7,10). Excess
production of free radicals, nitric oxide and superoxide is closely related to cell and tissue pathology
caused by mustard (3). There is neither an effective
treatment to toxic effects of mustards, nor a therapeutic antidote to them (5,6).
Proanthocyanidine (PC) is a free radical scavenger,
and at the same time it has anti-inflammatory effects
(11,12). The objective of the present study was to
investigate the role of oxidative stress status in BTT
* Department of Thoracic Surgery, Gulhane Military Medical Faculty
** Department of Histology and Embryology, Ankara University School of Medicine
*** Department of Neurosurgery, Ministry of Health, Ankara Training and Research Hospital
**** Department of Nuclear, Biologic and Chemical Warfare, Gulhane Military Medical Faculty
***** Department of Ophthalmology, Gulhane Military Medical Faculty
****** Department of Pharmaceutical Toxicology, Gulhane Military Medical Faculty
******* Department of Biological Sciences, Biotechnology Research Unit, Middle East Technical
University
Reprint request: Dr. Orhan Yücel, Department of Thoracic Surgery, Gulhane Military Medical
Faculty, Etlik-06018, Ankara, Turkey
E-mail: orhanycl@gmail.com
Date submitted: February 10, 2009 • Date accepted: March 25, 200940 • March 2009 • Gulhane Med J Yücel et al.
coexisting with mustard toxicity, and to determine
the protective effect of PC. Up to date there are several studies related to NM (1,3), but this is the first
study that reveals a developed war model in which
both trauma and chemical weapons are applied at the
same time, as in the case of real terror attacks.
Material and Methods
The study was performed in Gülhane Military
Medical Academy Animal Research Laboratory, and
it was approved by the Ethics Committee of Gülhane
Military Medical Academy.
Chemicals: NM and the chemicals required for the
oxidative stress analysis were obtained from Sigma–
Aldrich Chemie GmbH (Taufkirchen, Germany)
and the organic solvents were bought from Merck
KGaA (Darmstadt, Germany). A commercially available grape seed PC extract was purchased from GNC
Bakara A.Ş. (Proantosiyanidin: GN 6018, 100 mg, 90
capsules, Istanbul, TR).
Animals: Sixty adult Ratus Norvecus rats, weighing
160±10 grams were used. The rats were separated into
four groups by the simple random sampling method
and each group contained fifteen rats.
Experimental design: The first group was the control
group (CG): Vaporized 5 ml distilled water was applied for 10 minutes and the rats were not exposed to
trauma and NM. Subjects were sacrificed by anesthetic
with lethal dose after a 3-days follow up period. Two
samples were taken from the lungs, one of which was
fixed in 2.5% buffered glutaraldehyde for histopathological examination and the other was kept in liquid
nitrogen for biochemical analysis. MDA level, activities of superoxide dismutase (SOD), glutathion peroxidase (GPx) and catalase (CAT) were measured with
biochemical analyses in the lung tissues of sacrificed
subjects. With the method we mentioned briefly in
the following part of the paper, histopathological
and biochemical investigation were performed at the
tissue samples, and results have been recorded.
Histological examination: The lung tissue was removed, sectioned into small pieces and fixed in 2.5%
glutaraldehyde in 0.1 mol/l phosphate buffer, pH 7.2
at + 4 °C for 2-4 h and post-fixed in 1% osmium tetroxide in phosphate buffer (pH 7). Later the materials
were dehydrated in serially increasing concentrations
of alcohol. The tissues were then washed with propylene oxide and embedded in Araldite 6005 (CibaGeigy, Summit, NJ, USA). Semi-thin sections of 0.8
μm were cut with a glass knife on an ultramicrotome
Leica Ultracut R (Leica, Solms, Germany) stained with
toluidin blue azur II and then examined under a Zeiss
Axioscope photomicroscope (Thornwood, NY, USA).
Ultrathin sections of 60 nm were cut with a glass knife
on a Leica Ultracut R ultramicrotome, stained with
uranyl acetate and lead citrate and examined on a
LEO 906 E (LEO Elektronenmikroskopie, Oberkochen,
Germany) transmission electron microscope.
Oxidative stress status related parameter analysis
Tissue preparation for oxidative stress status: Tissue
samples were homogenized in 1.5% KCl solution on
ice using a glass homogenizer. Then homogenized
samples were centrifuged for 10 min at 5000xg and
4
o
C. Supernatant was used for the analysis.
GPx activity measurement: GPx activities in tissue
homogenates were measured by the method described in our previous studies (13,14). The reaction
mixture was 50 mmol/L tris buffer, pH 7.6 containing 1 mmol/L of Na
2
EDTA, 2 mmol/L of reduced glutathione (GSH), 0.2 mmol/L of NADPH, 4 mmol/L
of sodium azide and 1000 U of glutathione reductase (GR). Fifty μL of tissue homogenate and 950 μL
of reaction mixture were mixed and incubated for 5
min at 37
o
C. Then the reaction was initiated with
10 μL of t-butyl hydroperoxide (8 mmol/L) and the
decrease in NADPH absorbance was followed at 340
nm for 3 min. Enzyme activities were reported as U/g
in tissue.
MDA level measurement: MDA levels in tissue homogenate samples were determined in accordance
with the method described in our previous studies
(13,14). MDA levels were expressed as TBARS. After
the reaction of thiobarbituric acid with MDA, the
reaction product was measured spectrophotometrically. Tetramethoxy propane solution was used as
standard.
SOD activity measurement: CuZn-SOD activity in tissue
homogenate was measured by the method described in
our previous studies (13,14). Briefly, each homogenate
was diluted 1:400 with 10 mM phosphate buffer, pH
7.00. Twenty five L of diluted hemolysate was mixed
with 850 L of substrate solution containing 0.05 mmol/
L xanthine sodium and 0.025 mmol/L 2-(4—iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride
(INT) in a buffer solution containing 50 mmol/L CAPS
and 0.94 mmol/L EDTA pH 10.2. Then, 125 L of xanthine oxidase (80 U/L) was added to the mixture and
absorbance increase was followed at 505 nm for 3 min
against air. Twenty five L of phosphate buffer or 25 L
of various standard concentrations in place of sample
were used as blank or standard determinations. CuZnSOD activity was expressed in U/g tissue.
CAT activity measurement: CAT activity in tissue
homogenate was measured by the method of Aebi
(13,14). The reaction mixture was 50 mM phosphate Volume 51 • Issue 1 Proanthocyanidin and trauma model • 41
buffer pH 7.0, 10 mM H2
O2
and homogenate. The
reduction rate of H2
O2
was followed at 240 nm for 30
seconds at room temperature. Catalase activity was
expressed in U/g tissue.
The second group was the PC group (PCG): The same
protocol (three days follow up, vaporized distilled
water application, sacrification, histopathological
and biochemical sampling, histopathological and
biochemical evaluation and recording) was applied
with the control group. Differently from the CG,
PC administration (100 mg/kg body weight/oral/via
Gavage) has been started 8 hours before the three
days of follow up period onset. PC treatment continued until the subjects were sacrificed.
The third group was the traumatized NM group (TMG):
The same protocol (three days follow up, vaporized
distilled water application, sacrification, histopathological and biochemical sampling, histopathological
and biochemical evaluation and recording) was applied with the control group. Differently from the
CG, 1- BTT was applied before the three days of follow up period, 2- Traumatized subjects were exposed
to NM via vaporized distilled water. BTT and NM
were administrated in the ways we described in our
previous studies (13,15).
Blunt thorax trauma application: The BTT administration model that we developed before (GATA trauma
model) was administered to the subjects in order to
form a BTT. The BTT forming model is briefly is that:
Rats were anesthetized with intraperitoneal Ketamine
hydrocloride 90 mg/kg and Xylazine. After the anesthesia, rats were placed in right lateral decubitis
position over the GATA trauma model’s support part
(Figure 1). Forty grams metal weight (0.004 joule) was
used to form trauma on the subjects. Rats were placed
in lateral decubitis position over the trauma model’s
support part. Metal weights were dropped from 1 m
height onto rats through the plastic pipe for gaining
BTT. BTT was applied at the right lateral axial axe, on
the fourth intercostal space.
NM exposure: The subjects were exposed to NM via
the method we described in our previous studies. In
brief: after BTT application, the rats were placed in the
chamber. Rats were directly exposed to toxic dose of
vaporized 8 mg NM dissolved in 5 ml distilled water
for 10 minutes, 800 mg/m3
/min. All exposures were
performed in a 100 L volume chamber equipped with
chemical, biological, radiological and nuclear filters.
Fourth (treatment) group (TG): The same protocol
(three days follow up, BTT, NM exposure, sacrification, histopathological and biochemical sampling,
histopathological and biochemical evaluation and
recording) was applied with the TMG. Differently
from the TMG, PC administration (100 mg/kg body
weight/oral/via gavage) has been started 8 hours before BTT administration and NM exposure. PC treatment continued until the subjects were sacrificed.
Statistical analysis: Statistical analysis was performed
by using Kruskal-Wallis and Bonferroni-corrected
Mann-Whitney U tests. All the results were assessed
as the mean with min and max and p <0.05 was accepted as statistically significant.
Results
Results of histological examination: In CG, typical normal structural findings were seen in both light and
electron microscopic observations. The alveolar wall
consisted of surface epithelium, supporting tissue and
blood vessels. Most of the alveolar surface area was covered by large squamous type I pneumocytes which was
seen to have densely stained nuclei in histological section. Type II pneumocytes had large round nuclei with
a prominent nucleolus and vacuolated cytoplasm. It
was seen that capillary blood vessels had formed an extensive plexus around each alveolus. Alveolar macrophages could be found on the surface of alveolar lining
cells as well as in the supporting tissue of the alveolar
septa. Light micrographs and electronmicrographs of
TMG capillary dilatation and eythrocyte plugging in
capillaries were observed. The most striking change to
be noticed was numerous numbers of alveolar macrophages. Light micrographs of the PCG were normal in
appearance, similar to the CG findings. Furhermore,
light micrographs of the TG were normal in appearance, similar to the CG findings too. The abnormal appearance of the lung tissue in NM were mostly but not
completely corrected by PC. These findings can imFigure 1. Blunt thorax trauma model (GATA trauma model). Metal
weights (50 g) were dropped from 1 m height onto rats through the
plastic pipe for gaining blunt thorax trauma42 • March 2009 • Gulhane Med J Yücel et al.
ply that the structural changes induced by NM could
be partially prevented and restored by PC treatment.
Representative histopathological pictures of the study
groups are demonstrated in Figures 2 and 3.
MDA levels, and GPX, SOD and CAT activities in tissue: Oxidative stress status analysis included MDA
level, and SOD, CAT and GPx activities. MDA levels,
CAT and GPx activities in PCG were similar to CG.
NM direct exposure caused increased MDA levels,
and decreased GPx and SOD activity significantly in
lung tissue. PC treatment decreased MDA levels, but
CAT and GPx activities were similar to those of TMG
group. The levels of histochemical parameters according to the study groups are presented in Table 1.
Discussion
NM is the most widely used chemical agent in war
and terror attacks (1,16). It affects many organs such
as respiratory tract, eyes, skin, gastrointestinal and
central nervous systems (1,3). Because of their significant effects on respiratory system, mustards are
extremely relevant to trauma-dealing medical staff
too. In addition, mustards are a mutagenic, carcinogenic and cytotoxic agent (17,18). It has been shown
that mustard toxicity comes from oxidative as well as
nitrosative stress leading to lipid, protein and DNA
damage in the target cell (1). Yaren et al. have reported that peroxynitrite may be responsible, at least in
part for NM-induced lung toxicity, and peroxynitrite
scavengers may be useful in order to prevent mustard
toxicity (1). Excess production of free radicals, nitric
oxide and superoxide is closely related to cell and tissue pathology of mustard (3). NM exposure or BTT
also causes inflammatory lung diseases, including
acute respiratory distress syndrome (7,10).
Pulmonary contusion, parenchymal lung injury,
hemothorax and pneumothorax are the most common pulmonary injuries after BTT (19). Good surgical
Figure 2. a. Control group: Toluidin blue-Azur IIx250,
b. Proanthocyanidin group: Toluidin blue-Azur IIx250, c. Traumatized
mustard group: Toluidin blue-Azur IIx250, d. Treatment group:
Toluidin blue-Azur IIx250. *: capillary lumen, P1: type 1 pneumocyte,
P2: type 2 pneumocyte, arrowheads: alveolar macrophages
Figure 3. a. Control group x1000, b. Proanthocyanidin group x1000,
c. Traumatized mustard group: x2156, d. Treatment group: Gx1000,
e. Treatment group: x1000, f. Treatment group: x2156. For electron
microscopic examination all of the figures are stained with uranil
acetat-lead citrat. *: capillary lumen, P1: type 1 pneumocyte, P2: type
2 pneumocyte, arrowheads: alveolar macrophages, e: erythrocyte
Table I. Oxidative stress related parameters of the lung tissue after blunt thoracic trauma and mustard exposure and proanthocyanidin treatment in rats
Groups (n) Malondialdehyde (nmol/g)* Glutathione peroxidase (U/g)* Catalase (U/g)* Superoxide dismutase (U/g)*
Control group 15 6.87 (6.71/8.01) 45.07 (43.58/48.19) 3.16 (3.02/3.24) 240.20 (224.28/250.89)
Proanthocyanidin group 15 6.77 (6.69/6.82) 43.17 (42.95/44.92) 3.36 (3.28/3.45) 247.24 (235.81/258.14)
Nitrogen mustard group 15 9.37 (9.24/9.48)# 28.85 (27.01/29.98) # 3.26 (3.16/3.75) 92.03 (85.02/94.44)#
Treatment group 15 7.07 (7.01/7.22)Φ 28.79 (26.51/30.14) 3.18 (3.01/3.22) 113.88(107.08/125.88)
*: Values are given as mean (min/max)
#: p<0.001, Nitrogen mustard group compared with control group
Φ: p<0.05, Treatment group compared with nitrogen mustard groupVolume 51 • Issue 1 Proanthocyanidin and trauma model • 43
outcomes are reported about management of these
pulmonary injuries, and in general, vascular repair,
pneumoraphy and chest tube thoracostomy are sufficient treating procedures for them (19,20).
In contrast, pulmonary contusion is a challenge
for a thoracic surgeon and intensive care specialist.
Pulmonary contusion represents a spectrum of lung
injury characterized by the development of paranchymal infiltrates and various degrees of respiratory
dysfunction. There is a spectrum of injury severity,
ranging from localized consolidation with little clinical impact, to acute lung injury and ARDS (20-22).
Currently, it is believed that pulmonary contusion is
the most common potentially life-threatening pulmonary injury (20,22).
As we demonstrated, NM and BTT have individually important hazardous effects on lungs. It is easy
to predict that patients with blunt traumatized lungs
after NM exposure will have increased mortality and
morbidity rates. There are numerous experimental
studies about BTT and NM (1,3,23-26). However, to
our knowledge there is no experimental study concerning BTT and chemical weapons effects together.
In this study we simulated the scenario of a terror
attack to patients with blunt traumatized lungs after
chemical gas exposure.
There is still no beneficial treatment or therapeutic
antidote available to toxic effects of mustards (5,27).
In addition, pulmonary contusion treatment is generally supportive (20). There are ongoing researches
into such agents that may have antioxidant properties (28,29). It is essential that further thorough investigation is done in this area.
It has been revealed that PC is a free radical scavenger
and it has also anti-thrombotic and anti-inflammatory
effects (11,12,30,31). It has been shown that, in addition to its antioxidative property, it enhances low-level
production of intracellular NO in primary rat astroglial
cultures. Moreover, PC pretreatment protects the microglial GSH pool during high output NO production
and results in an elevation of the H2
O2
tolerance in
astroglial cells (17). It has been stated that IH636 GSPE
provides superior antioxidant efficacy as compared to
Vitamins C, E and -carotene (13). It is clear that novel
antioxidants have sufficient effects against free radicals and cardiovascular disease to provide organism.
(13,31). In particular, novel antioxidants can neutralize harmful free radicals and their damaging effects
on tissue and organs as well as increasing the body’s
antioxidant status (13). PC is a combination of biologically active polyphenolic flavonoids including oligomeric PCs. Their biological, pharmacological, therapeutic, and chemoprotective properties against oxidative stress and oxygen free radicals have already been
demonstrated (13,31). PC is a potent bioavailable free
radical scavenger that provides significant protection
over multiple target organs against structurally diverse
drug and chemically induced toxic manifestations in
rats (13). The mechanistic pathways performed by PC
to provide cardioprotectin includes: (a) potent hydroxyl and other free radical scavenging abilities, (b)
anti-apoptotic, anti-necrotic and anti-endonucleolytic
potentials, (c) modulatory effect on apoptotic regulatory bcl-XL, p53 and c-myc genes, (d) cytochrome
P450 2E1 inhibitory activity, (e) inhibitory effects on
adhesion molecules, (f) modulatory effects on proapoptotic and cardioregulatory genes c-JUN, JNK-1, and
CD36 (13,30,32,33).
The histological examination and oxidative stress
status related parameters of our study showed similar findings in PCG and CG. These findings support
that PC has no unpredicted side effects. Furthermore,
due to its obvious antioxidant effect, PC can be an
efficient protector against blunt traumatized lungs
after chemical gas exposure. PC decreased MDA levels when compared to non-PC given group. However
GPX and CAT activities were not significantly different between PCG and TMG groups. In addition,
increased CAT activities were reported in PCG compared with TMG group (p<0.05). This demonstrated
that free radicals were scavenged by PC but SOD, CAT
and GPx activities were still lower than CG. It can
be concluded that free radicals were not removed
completely.
In this study light micrographs and electronmicrograps of TMG capillary dilatation and eythrocyte
plugging in capillaries were observed. The most striking change was numerous numbers of alveolar macrophages. PC treatment decreased histopathological
changes in TG. Light micrographs of the TG were
normal in appearance, similar to the CG findings.
The abnormal appearance of the lung tissue in TMG
were not completely but mostly corrected by PC. It
was shown that free oxygen radial damage has an important role in mustard toxicity.
These findings can imply that the structural changes induced by blunt traumatized lungs after NM exposure can be partially prevented and restored by PC
treatment. PC is a strong free oxygen radial scavenger
and because of this property it can be an option for
decreasing toxic patients‘ mortalities and morbidities.
Further clinic and experimental studies are needed to
prove PC and other antioxidant agent’s probable protective roles against mustard toxicity.