Proantosiyanidin nitrojen mustarda bağlı akciğer hasarını azaltır

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Introduction
One of the most important vesicant agents is mustard (1,2). It was first used in the battlefield during
World War I by Germany (1,3). Sulfur mustard (SM)
has posed a military threat and this is still considered
as a major danger against humankind (3,4). Both SM
and nitrogen mustard (NM) are known for their toxic
effects (5). NM is a structural analogue of the SM (5).
NM has become the prototypical cancer chemotherapeutic compound and has remained the standard
compound for this purpose for many years (3,5). The
most destructive properties of mustards are assumed
to be upon respiratory system, eyes and skin (3,6,7).
Mustard has many biological actions, but the cytotoxic mechanism of mustard has not been fully clarified (3-5). However, biological damage from mustard
may result in DNA alkylation, cross linking of DNA,
activation of proteases, resulting in proteolysis of several important enzymes and structural proteins, production of free radicals and induction of free radical
mediated oxidative stress, inflammation, activation
of TNF-α, part of the inflammatory cytokine (2,4,6-
8). This leads to cellular death and inflammatory reaction (3,5,6). Mustard gas exposure also causes inflammatory lung diseases, including acute respiratory
distress syndrome (6,7).
It is well known that proanthocyanidine (PC) is
a free radical scavenger that has anti-inflammatory
and anti-thrombotic effects (9,10). The grape seed
PC extract contains 54% dimeric PCs, 13% trimeric
PCs, 7% tetrameric PC, small amounts of monomeric, high-molecular-weight oligomeric PCs and flavonoids. Bagchi et al. have stated that grape seed extract
containing PC provides superior antioxidant efficacy
as compared to Vitamins C, E and β-carotene (11).
Novel antioxidants may offer an effective and safe
means of counteracting some of the problems and
bolstering the (organism) body’s defenses against free
radicals and cardiovascular disease (11,12). Based on
* Department of Thoracic Surgery, Gulhane Military Medical Faculty
** Department of Pathology, Gulhane Military Medical Faculty
*** Department of Pharmaceutical Toxicology, Gulhane Military Medical Faculty
**** Department of Cardiovascular Surgery, Gulhane Military Medical Faculty
***** Department of Nuclear, Biologic and Chemical Warfare, Gulhane Military
Medical Faculty
Reprint request: Dr. Orhan Yücel, Department of Thoracic Surgery,
Gulhane Military Medical Faculty, Etlik-06018, Ankara, Turkey
E-mail: orhanycl@gmail.com
Date submitted: November 27, 2008 • Date accepted: December 21, 2008268 • December 2008 • Gulhane Med J Yücel et al.
this preventive effect in these experimental studies,
we aimed to investigate the possible beneficial protective effects of PC in mustard toxicity.
NM is inexpensive, easily obtainable and frequently stockpiled. Because of those properties it has been
used as a chemical warfare agent in conflicts and terrorist attacks. In recent years, a lot of experimental
studies have been designed to investigate the cytotoxic mechanism induced by mustard and initial
event leading to cell death. The aim of this study
was to investigate the role of oxidative stress status
in mustard toxicity and to determine the protective
effect of PC which is a potent free radical scavenger
in mustard toxicity.
Material and Methods
Animals: The study was performed in the Animal
Research Laboratory and was approved by the Ethics
Committee. Forty five adult Ratus Norvecus weighing between 140 and 160 g were used. The rats were
separated into three groups by the “simple random
sampling method” and each group contained fifteen
rats.
Chemicals: NM and chemicals for oxidative stress
related analysis were provided from Sigma–Aldrich
Chemie GmbH (Taufkirchen, Germany) and organic
solvents from Merck KGaA (Darmstadt, Germany). A
commercially available PC was purchased from GNC
Bakara A.S. (Proanthocyanidine: GN 6018, 100 mg,
90 capsules, Istanbul, Türkiye).
Experimental design: In experimental study, animals
were divided into three groups. The first group was
control group (CG) which was only exposed to vaporized 5 ml distilled water for 10 minutes. The second
group was NM group (NMG) which was exposed to
a toxic dose of vaporized 8 mg NM dissolved in 5 ml
distilled water for 10 minutes (800 mg/m3
/minutes).
The third group was PC group (PCG) and PC containing diet (1X100+/-5 mg/kg rat body weight/day)
was given orally by gavage to this group. PC intake
in this group was started 8 h before being exposed
to the same dose of NM as in the NMG and continued for three days. All exposures were performed in
a 100 L volume chamber equipped with Chemical,
Biological, Radiological, and Nuclear (CBRN) filters.
The procedure: Rats were anesthetized with intraperitoneal Ketamine hydrocloride (Ketamine hydrochloride solution in 5%, Parke – Davis licensed
Eczacıbaşı Medical Industry, Istanbul) 90 mg/kg and
Xylazine (Xylazine solution in 2%, by Parke – Davis
licensed Eczacıbaşı Medical Industry, Istanbul, 10
mg/kg). Rats were placed in a chamber. All rats
were heated (average temperature: 22±2
0
C) by using tungsten electric bulb (100W/220V) and oxygen
was supplied during the procedure. Rats were directly exposed to vaporized nitrogen mustard. Analgesia
was obtained by using buterfenol (0.5 mg/kg, s.c.).
In PCG, PC intake was started 8 h before NM application and continued 72 hour by gavage with a
commercially available IH636 grape seed PC extract.
In NMG, homogeneous methylcellulose solution
intake was started 8 h before NM application and
continued 72 h by gavage without a grape seed PC
extract. Grape seed PC was homogenized in 2 mL
1% methylcellulose solution and then diluted with
0.9% NaCl to 10 mL (12). The rats were given 100±5
mg/kg of the grape seed PCs in the form of an extract
(11). Thereafter, the animals were allowed to survive
for an additional 72 hour. All rats were sacrificed after 72nd hour by giving lethal dose of xylazine and
ketamine. Their chests were opened by median sternotomy. The lungs were removed and fixed in 10%
buffered formalin solution immediately by intratracheal instillation for histopathological evaluation.
One third of upper lobes of both lungs were kept
in liquid nitrogen for oxidative stress status analysis
before fixation.
Tissue preparation for histopathological evaluation:
Both right and left lungs for all cases were evaluated
for histopathological examination. After routine tissue processing, 4-μm thick sections from each formalin-fixed paraffin-embedded tissue were stained with
haematoxylin and eosin (H&E). H&E stained slides
were reviewed by two pathologists. Histological parameters including alveolar edema, alveolar congestion, alveolar hemorrhage, interstitial inflammation
and bronchiolocentric inflammation were assessed
semi-quantitatively using a 4-stage grading scale:
negative (-), weakly positive (1+), moderately positive (2+), strongly positive (3+).
Oxidative stress status related parameter analysis
Tissue preparation for oxidative stress status: Tissue
samples were homogenized in 0.2 mMol (pH 7.5)
KCl buffer 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 (5,13-15).
Glutathione peroxidase (GSH-Px) activity measurement: GSH-Px activities in plasma samples and tissue homogenates were measured by the method
described in our previous study (13). The reaction
mixture was 50 mMol tris buffer, pH 7.6 containing
1 mMol of Na2EDTA, 2 mMol of reduced glutathione (GSH), 0.2 mMol of NADPH, 4 mMol of sodium azide and 1000 U of glutathione reductase (GR).
Fifty μL of plasma or tissue homogenate and 950 μL Volume 50 • Issue 4 Proanthocyanidine and lung damage • 269
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) and the
decrease in NADPH absorbance was fallowed at 340
nm for 3 min. Enzyme activities were reported as
u/mg in tissue.
Malondialdehyde (MDA) level measurement: MDA
levels in plasma and tissue homogenate samples
were determined in accordance with the method described in our previous study (14). MDA levels were
expressed as Thiobarbituric Acid Reactive Substances
(TBARS). After the reaction of thiobarbituric acid
with MDA, the reaction product was measured spectrophotometrically. Tetramethoxy propane solution
was used as a standard.
Superoxide dismutase (SOD) activity measurement: CuZn-SOD activity in tissue homogenate was
measured by the method as described previously (5).
Briefly, each homogenate was diluted 1:400 with
10 mM phosphate buffer, pH 7.00. 25 μL of diluted
hemolysate was mixed with 850 μL of substrate solution containing 0.05 mMol xanthine sodium and
0.025 mmol/L 2-(4-iodophenyl)-3-(4-nitrophenol)-
5-phenyltetrazolium chloride (INT) in a buffer solution containing 50 mMol CAPS and 0.94 mMol EDTA
pH 10.2. Then, 125 μL of xanthine oxidase (80 U/L)
was added to the mixture and absorbance increase
was followed at 505 nm for 3 minutes against air.
25 μL of phosphate buffer or 25 μL of various standard concentrations in place of sample were used as
blank or standard determinations. CuZn-SOD activity was expressed in U/mg tissue.
Catalase (CAT) activity measurement: CAT activity in
tissue homogenate was measured by the method of
Aebi (15). The reaction mixture was 50 mMol phosphate buffer pH 7.0, 10 mMol 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/mg tissue.
Statistical analysis was done to analyze the two
groups mutually by using Kruskal-Wallis and MannWhitney U tests. The results were expressed as the
median (min–max), and p<0.05 was assessed as statistically significant.
Results
Histopathological evaluation: Representative histopathological pictures of the study groups are demonstrated in Figure 1. Control animals were presented with normal lung tissue. Alveolar edema, parenchymal congestion, alveolar hemorrhage, interstitial
inflammation and bronchiolocentric inflammation
were not encountered in the lungs of the control
cases. Histopathological evaluation of rat lungs following NM administration revealed that the development of lesions predominated in the parenchymal
region. The lung lesions following NM administration were alveolar edema, parenchymal congestion,
Figure 1. Histopathological examination of the control group shows normal pulmonary parenchyma (a H&Ex40 and b H&Ex200). One of the
cases in NMG group showed dense interstitial inflammation, alveolar collapse, parenchymal congestion and minimal alveolar hemorrhage in
the lung parenchyma (c H&Ex40 and d H&Ex200). In the treatment group, a case demonstrated mild interstitial inflammation (e H&Ex40 and
f H&Ex200)270 • December 2008 • Gulhane Med J Yücel et al.
alveolar hemorrhage, interstitial inflammation and
bronchiolocentric inflammation. In the lungs of PCtreated rats, alveolar edema, alveolar hemorrhage,
and inflammatory cell infiltration and airways pathology were also observed (Table I).
There were severe alveolar congestion, interstitial
inflammation and bronchial inflammation and many
of the alveoli obstruction in NMG. Inflammatory
cells were also apparent around bronchial mucosal
epithelium and interstitial tissue. Many airways were
collapsed. In addition, significant interstitial inflammation was also observed after PC treatment. Lung
parenchyma showed inflammation into the airways
and alveoli. In the PC treated rats significant less alveolar congestion, alveolar hemorrhage, and airways
pathology was observed. The degree of histopathological parameters according to study groups is presented in Table I.
MDA levels, and GSH-Px, SOD and CAT activities in
tissue: Oxidative stress analysis included MDA level,
and SOD, CAT and GSH-Px activity. NM direct exposure caused significantly higher MDA levels, and
lower GSH-Px, SOD and CAT activity in lung tissue.
On the other hand, PC treatment decreased MDA levels, and SOD, CAT and GSH-Px activities were similar
to those of NMG group. (Table II)
Discussion
Mustard, one of the most important vesicant
agents, affects respiratory system and causes some
degree of damage on other tissue and organs (5,6,16).
Mustards were also reported as a mutagenic, carcinogenic, and cytotoxic agent (16). Current knowledge
makes it seem feasible that mustard toxicity comes
from oxidative as well as nitrosative stress leading
to lipid, protein and DNA damage in the target cell
(5). Yaren et al. showed that scavenging peroxynitrite
and inhibiting iNOS have similar protective effects.
They thought 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 (5). In our study, NM direct exposure caused increased MDA levels, and significantly decreased GSH-Px, SOD and CAT activity in lung
tissue. These findings suggest that free oxygen radial
damage has an important role in mustard toxicity.
It is well known that PC is a free radical scavenger
and it has also anti-thrombotic and anti-inflammatory effects (9,10,17,18). In particular, novel antioxidants can neutralize harmful free radicals and
their damaging effects on tissue and organ as well
as enhancing the body’s antioxidant status (11).
Grape seed PCs, a combination of biologically active
Table I. Histopathological results of lung tissue after mustard exposure and proanthocyanidine treatment in rats
Groups n Alveolar edema Parenchymal
congestion
Alveolar hemorrhage Interstitial
inflammation
Bronchiolocentric
inflammation
- 1+ 2+ 3+ - 1+ 2+ 3+ - 1+ 2+ 3+ - 1+ 2+ 3+ - 1+ 2+ 3+
Control group 15 15 0 0 0 15 0 0 0 15 0 0 0 15 0 0 0 15 0 0 0
Nitrogen mustard-only group 15 14 1 0 0 3* 7 4 1 9* 3 2 1 8* 4 3 0 11* 2 1 1
Nitrogen mustard plus
proanthocyanidine group
15 15 0 0 0 6** 6 3 0 12** 2 1 0 6** 5 3 1 12** 2 1 0
*: p<0.001, Nitrogen mustard-only group compared with control group
**: p<0.05, Nitrogen mustard plus proanthocyanidine group compared with nitrogen mustard-only group
Table II. Oxidative stress related parameters of the lung tissue after mustard exposure and proanthocyanidine treatment in rats
Groups n Malondialdehyde
(nmol/mg)*
Glutathione peroxidase
(U/mg)*
Catalase
(U/mg)*
Superoxide dismutase
(U/mg)*
Control group 15 6.87±0.35 0.045±0.0058 0.031±0.0040 0.240±0.030
Nitrogen mustard-only group 15 8.05±0.70** 0.023±0.002** 0.002±0.0004** 0.117±0.040**
Nitrogen mustard plus
proanthocyanidine group
15 7.25±0.85*** 0.024±0.004 0.002±0.0016 0.102±0.063***
*: Values are given as mean±standard deviation
**: p<0.001, Nitrogen mustard-only group compared with control group
***: p<0.05, Nitrogen mustard plus proanthocyanidine group compared with nitrogen mustard-only groupVolume 50 • Issue 4 Proanthocyanidine and lung damage • 271
polyphenolic flavonoids including oligomeric PCs,
have been demonstrated to exert a novel spectrum
of biological and therapeutic properties against oxidative stress and oxygen free radicals (11,18). Bagchi
et al. have demonstrated that GSPE is a potent bioavailable scavenger of free radicals that provides significant protection towards multiple target organs
against structurally diverse drug and chemically induced toxic manifestations in rat (100 mg/kg body
weight, p.o.) (11). Our study showed that due to its
obvious antioxidant effect proanthocyanidine can
be an efficient protector against NM. PC treatment
decreased MDA levels when compared to non-PC
given NMG group. However GSH-Px, SOD and CAT
activities were not significantly different in PCG
and NMG group. This showed that free radicals were
scavenged by PC but SOD, CAT and GSH-Px activities were still lower than CG. It can be concluded
that free radicals were not removed completely.
Previous studies have exhibited that the primary
airway lesion from mustard is necrosis of the mucosa with later damage to the musculature of the
airways (3,4,6). In our study, only one rat had hemorrhagic pulmonary edema in NMG.
Inhalation of mustard gas incites acute respiratory
distress syndrome (ARDS) due to hemorrhagic inflammation (6,7). Most deaths are due to secondary
respiratory infection (6). Death often occurs between
the fifth and tenth day after exposure because of
pulmonary insufficiency and infection complicated
by a compromised immune response from agent-induced bone marrow damage (3,4). In this study it
was found that the main NM-related airway lesions
were alveolar edema, alveolar congestion, alveolar
hemorrhage, interstitial inflammation and bronchiocentric inflammation.
Right at this point, the major subject we were
strongly interested was the level and adequacy of
the lung damage. It was because the way we used to
induce that damage. It is obvious that many studies about lung damage induced by NM have already
been done in time and the results were sufficient.
But in none of these studies, rats were directly exposed to vaporized nitrogen mustard; usually they
have been exposed to NM solutions intratracheally
(1,5). Our exposure method was set to simulate the
war field as similar as it can be.
Histopathological evaluation confirmed lung
damage with NM exposure in NMG. As these findings were parallel and very similar to the findings of
former studies about the same issue, they brouht up
the adequacy and appropriateness of the method we
used to induce a lung damage related to NM (1,5).
PC treatment decreased histopathological changes
in PCG. It was shown that free oxygen radial damage has an important role in mustard toxicity. PC,
which is a strong free oxygen radical scavenger, has
an acceptable effect for decreasing toxic outcomes.
Further clinic and experimental studies, with the
same and/or other antioxidant agents, could prove
their probable protective roles against mustard
toxicity.
Indeed, this study includes many variable parameters i.e; exposed NM dose, exposure period, proanthociyanidine administration dose, treatment onset time, etc. Thus for a better treatment protocol,
further studies are needed to be done about these
parameters.