Chapter 5
Histological, Molecular and Biochemical
Studies of the Lung and Liver of Mouse
Exposed to Acute Cigarette Smoke
Histological, Molecular and Biochemical Study of Mouse Exposed to Acute Cigarette Smoke
5.1 Introduction
Cigarette smoke (CS) induced emphysema is one of the major causes of COPD
(Spurzem & Rennard, 2005; Belvisi & Bottomley, 2003). CS is a complex
mixture of at least 4000 different carcinogens that include nicotine, nitrosamine,
polycyclic aromatic hydrocarbons (PAH), aromatic amines, unsaturated aldehydes
(e.g. crotonaldehyde) and some phenolic compounds which mediate tumor
initiation and promotion (Palmer et al, 2005). Benzo-a-pyrene (B(a)P), a member
of PAH is a well-known procarcinogen, which metabolized through detoxification
pathway, however, its metabolic activation leads to formation of reactive
intermediates and potentially damaging metabolites that can promote cell injury
and elicit toxic effects (Miller and Ramos, 2001). The enzymes involved, include
notably phase I metabolic enzymes i.e. CYP1A1, mEPHX, which can also be
considered as phase II enzyme, and phase II conjugating enzymes i.e. GSTs
(Miller and Ramos, 2001). The metabolic activation of B(a)P to various reactive
intermediates, including epoxides, phenols, and quinones, is catalyzed by the
CYP1A1-containing mixed function oxidase system (Omiecinski et al, 1999).
Subsequent metabolic step may involve the hydration of epoxides to dihydrodiols,
mediated by the mEPHX, which may be followed by further oxidation of these
metabolites by CYP1A1 to form highly electrophilic and mutagenic B(a)P diol
epoxides (BPDEs) (Omiecinski et al, 2000). Several of the reactive intermediates
arising during the metabolism of B(a)P have been shown to be conjugated with
glutathione (Eaton and Bammler, 1999).
The gas phase of CS contains free radicals such as superoxide radicals,
hydroxyl radicals and hydrogen peroxide (H2O2) (Leone A, 2003; Vayssier et
al, 1998) that are known to activate redox sensitive transcription factors
(Nishikawa et al, 1999; Manna et al, 2005). Oxidative effects via free radical
generation in smokers cause LPO, oxidation of proteins and damage to tissues
mainly that of lung. The antioxidant enzymes superoxide dismutase (SOD) and
glutathione peroxidase (GPx) are also severely affected by CS resulting in
deleterious effects (Ozguner et al, 2005).
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CS also induces DNA single strand breaks (SSB) in human cells in vitro, and
those are attributed to free radical generation from cigarette smoke (Leanderson
and Tagesson, 1992; Nakayama et al, 1985; Spencer et al, 1995). Inhaled cigarette
smoke causes in vivo DNA-SSB in lung and liver of mice (Villard et al, 1998).
In this study, the investigation undertaken whether, acute-term CS exposure to mice
leads to histological changes in lungs and livers. Beside that we have done comet
assay in the erythrocytes to reveal CS induced DNA damage. Another aim was to
investigate the acute-term CS-induced oxidant/antioxidant imbalances related to
oxidative stress. We have also examined the possible influence of CS on the induction
of CS-metabolizing enzymes in lungs and livers. Therefore, the work was undertaken
to determine the expression of detoxifying genes like CYP1A2, mEPHX, GSTP1 and
oxidative stress related gene CYBA at RNA and protein levels in lungs and livers of
acute-term CS exposed mice. A known antioxidant, N-acetyl cysteine (NAC) was
also investigated for its potential to reduce acute-term CS induced oxidative stress.
Accordingly, mice were exposed to inhalation exposure of acute term CS exposure
for 15 days and NAC instilled subcutaneously.
5.2 Materials and Methods
5.2.1 Animals
Seven week-old pathogen-free Swiss albino female mice weighing 25-30 g were
obtained from Hamdard University, New Delhi, India. The animals were housed
in polypropylene cages under controlled conditions of a 12-h dark/12-h light cycle
at temperature 25±2οC throughout the acclimatization period of one week. They
were fed with a standard commercial pellet diet and water ad libitum. All
experiments were conducted in accordance with institute’s guidelines provided by
the Animal Ethics Committee, which has approved the study.
5.2.2 Acute Cigarette Exposure
Three groups of mice (5 mice per group) were marked as control (C), cigarette
smoke treated (S) and the antioxidant NAC plus cigarette smoke treated (S+A).
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Histological, Molecular and Biochemical Study of Mouse Exposed to Acute Cigarette Smoke
Each group of mice except control group was placed in the inhalation chamber (40
cm long, 30 cm wide and 25 cm high), inside an exhaustion chapel. Acute
cigarette exposure was given to S group as 10 cigarettes burning in smoke
inhalation chamber simultaneously for a period of 2 hours/day daily for 15 days.
The S+A group was subcutaneously injected with 9 mg (300mg/kg, body weight)
of NAC suspended in sterile saline, prior to smoke exposure similar to S group.
Each cigarette was puffed 15 times for 3 min at a rate of 5 puffs/min. One puff
meant drawing 35 mL of cigarette smoke into a 50 mL syringe, and then blowing
this cigarette smoke. The inhalation chamber was opened, by removing its cover,
and the smoke evacuated for 2 min by exhaustion of the chapel. Fresh air
inhalation was performed for 2 min after every 10 min of cigarette exposure.
5.2.3 Histological Observation
Animals were sacrificed by cervical dislocation and necropsied for retrieval of
lungs and livers from each group. The tissues were fixed in formalin (pH 7.2),
processed for paraffin embedding and five micron sections stained with
Haematoxylin and Eosin stains for microscopy. Stained sections were evaluated
on a Labcon compound microscope with 10x and 40x objectives and digital
photographs taken with a Canon photomicrography system.
5.2.4 Comet Assay
The alkaline comet assay was performed as described by Singh et al. (1988) and
Shimazak (1999). Briefly, cell suspension (erythrocytes) of each group were
mixed in 1:10 with prewarmed 0.75% ultra-low gelling agarose (BDH Electran
44415 2G; gelling temperature 17°C) and layered on microscopic slides precoated
with 0.1% agarose on microscopic slides. After incubation at 4°C to allow the
formation of agarose gel, the slides were immersed in precooled lysis buffer (2.5
M NaCl, 100 mM EDTA, 300 mM NaOH, 10 mM Tris, 34 mM Nlauroylsarcosine, pH 10; 10% DMSO, 1% Triton X-100 were added just before
use) for 1 h at 4°C in the dark. Slides were put in a submarine-type electrophoresis
tank containing 300 mM NaOH, 1 mM EDTA (pH 13.5) for 15 min.
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Electrophoresis was then carried out at 1 V/cm for 10 min at 10°C. Slides were
rinsed 3 times with neutralization buffer (0.5 mol/L Tris, pH 7.4). After a brief
rinse in distilled water, slides were air dried at 45°C on a hot plate and stored in a
cool humid box until use. Comets were stained with propidium iodide (10 μg/ml
in phosphate buffered saline) after rehydration of slides in distilled water for 5
min and observed under an Olympus BX60 fluorescence microscope (Olympus
Optical Co., Tokyo, Japan).
5.2.5 RNA Isolation
Total RNA was isolated immediately from dissected lungs and livers of all the
mice of the three groups using TRIzol reagent kit (Invitrogen, USA). The RNA
pellet was washed with 70% ethanol and was stored at –80°C in diethylpyrocarbonate (DEPC) treated water. The concentration was quantified as
absorbance at 260 nm. Purity of RNA was checked by determining the
A260/A280 ratio. Absorbance was taken on Varian Cary 400 UV-visible
spectrophotometer and gel documentation was performed using BioRad Gel Doc
2000 to check the quantity and quality of the total RNA. The integrity of the
isolated RNA was tested on an agarose gel.
5.2.5.1 Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
The variant specific expression of GSTP1, mEPHX, CYBA, CYP1A1, CYP1A2,
and GSTM1 mRNA was investigated by two-step semi-quantitative RT-PCR.
Single strand cDNA was reverse transcribed using First-Strand cDNA Synthesis
kit (Amersham Biosciences, UK). Total RNA, 2 μg in 8 μl of DEPC treated water,
was heated at 65 °C for 10 min, chilled on ice and 5 μl of the Bulk First-Strand
cDNA reaction mix was added along with 1 μl of DTT solution and 1 μl of
oligod(T)18 primer provided in the kit. The contents were mixed and incubated at
37°C for 1 h. Primers for RT-PCR were carefully designed by Primer select
software. Primer details and amplification conditions of PCRs are provided in the
Table 5.1. RT-PCR for β-actin was performed for normalization. After gel
electrophoresis and ethidium bromide intercalation, PCR-amplified products were
visualized under UV light and analyzed by a computerized densitometry system.
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5.2.6 Western Blotting
Portions of liver and lung tissues were grind in liquid nitrogen and sonicated in
lysis buffer (7M urea, 2M thiourea, 2% DTT, 4% CHAPS and 0.8% pharmalyte
pH 3-10 cocktail, 50 mg of tissue per 200 μl of lysis buffer) and centrifuged to
obtain supernatant. The protein content in the supernatant was determined by
Bradford assay (Bio-Rad Laboratories, CA). 50 μg of each protein sample was
loaded and resolved on 12% SDS–PAGE and transferred to Hybond ECL
membrane (Amersham Bioscience). The membrane was blocked with 5% (w/v)
nonfat milk at room temperature for 2 h and incubated with mouse monoclonal
antibody against mEPHX (1:250, BD Biosciences, USA) and GSTP1 (1:1000, BD
Biosciences, USA) at room temperature for 1 h. After washing with phosphate
buffered saline with 0.1% Tween-20 (PBST), the membrane was incubated with
horseradish peroxidase-conjugated anti-mouse antibody (1:10000, Sigma, USA) at
room temperature for 1 h. After washing in PBS-T, the bands were visualized with
DAB (3,3’-Diaminobenzidine, Sigma) in accordance with the manufacturer’s
instructions.
5.2.7 Biochemical Characterization of Smoke Exposed Liver and Lungs
5.2.7.1 Preparation of Tissue Samples
Portions of liver and lungs were rinsed in ice cold PBS (pH 7.4), weighed and a
10% (w/v) tissue homogenate was prepared in ice-cold phosphate buffer (0.1 M,
pH 7.4). The homogenate was centrifuged and the resulting supernatant was used
for all the bioassays. Protein content was quantified by Bradford assay (Bio-Rad
Laboratories, CA) with BSA as the standard. All the assays were performed on
SpectraMax Plus 384 spectrophotometer (Molecular Devices, USA). Intra- and
inter-assay coefficient of variations were less than 10%.
5.2.7.2 Estimation of MDA and GSH Level
MDA and GSH level were assayed by the method of Wright et al (1981) and
Jollow et al (1974), respectively, as described in detail in Chapter 2.
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5.2.7.3 Estimation of Enzyme Activities
5.2.7.3.1 Superoxide Dismutase (SOD)
SOD activity was assayed according to the method of Marklund and Marklund.
Changes in the absorbance were recorded at 420 nm. In brief, the inhibition of
autoxidation of pyrogallol was measured as a function of time. The amount of
enzyme required to obtain 50% inhibition was considered equivalent to one unit of
SOD activity.
5.2.7.3.2 Catalase (CAT)
CAT activity was assayed by the method of Claiborne (1985) as described in
detail in Chapter 2.
5.2.7.3.3 Glutathione Peroxidase (GPx)
GPx activity was assayed by the method of Mohandas et al (1984) as described in
detail in Chapter 2.
5.2.7.3.4 Glutathione Reductase (GRx)
GRx activity was assayed by the method of Mohandas et al (1984). GRx activity
was quantitated by measuring the disappearance of NADPH at 340 nm and was
calculated as nmol NADPH oxidized/min/mg protein using the molar extinction
coefficient of 6.22X103 m−1cm−1.
5.2.7.3.5 Glutathione S-transferase (GST)
GST activity was assayed by the method of Habig et al (1974). The changes in
absorbance were recorded at 340 nm and enzyme activity was calculated as nmol
CDNB conjugate formed/min/mg protein using the molar extinction coefficient of
9.6X103 m−1cm−1.
5.2.8 Statistics and Data Presentation
All data are expressed as mean ± standard deviation (SD). Data were analyzed by
two-way analysis of variance (ANOVA). Data from S or S+A group were compared
with those of C group animals. Moreover, data from S+A group were compared
with those of S group, alone. The level of p < 0.05 was considered significant.
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Table 5.1: Primers and cycling conditions used for RT-PCR of GSTP1, mEPHX, CYBA, GSTM1 and CYP1A2 genes
S. No.
Gene
Primer pairs
Cycling conditions
1.
GSTP1
F 5’-CCA GTT CGA GGG CGG TGT GAG-3’
I 94°C 4', D 94°C 30'',
R 5’-CCA AAG AGC GGC CAA GGT GTC-3’
A 61°C 45'', E 72°C 45'',
30 cy, FE 72°C 10'
2.
mEPHX
F 5’-CGG TGG CCA CTG CGA GGA TC-3’
I 94°C 4', D 94°C 30'',
R 5’-CCA GGC CCA CAG GAG AGT CAT T-3’
A 63°C 45'', E 72°C 45'',
30 cy, FE 72°C 10'
3.
CYBA
F 5’-GCT GCC CTC CAC TTC CTG TTG T-3’
I 94°C 4', D 94°C 30'',
R 5’-GGC TGC CTC CTC TTC ACC CTC-3’
A 63°C 45'', E 72°C 45'',
30 cy, FE 72°C 10'
4.
GSTM
F 5’-GGA GGG ACC CGC TGT TTT GTC-3’,
I 94°C 4', D 94°C 30'',
R 5’-GGA TGG CAT TGC TCT GGG TGA T-3’
A 63°C 45'', E 72°C 45'',
30 cy, FE 72°C 10'
5.
CYP1A2
F 5’-CCC AAC CCG GCC CTC AAG A-3’
I 94°C 4', D 94°C 30'',
R 5’-TTG CCG ATC CCT GCC AAC CA-3’
A 61°C 45'', E 72°C 45'',
30 cy, FE 72°C 10'
6.
β-actin
F 5’-TTG CTG ACA GGA TGC AGA AGG -3’
I 94°C 4', D 94°C 30'',
R 5’-GCT GAT CCA CAT CTG CTG GAA-3’
A 66°C 30'', E 72°C 30'',
30 cy, FE 72°C 10'
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5.3 Results
5.3.1 Histological Features of Lung and Liver
Compound light microscopic observation of the sections of lung showed
remarkable difference among C, S and S+A groups (Fig. 5.1). The C group
showed normal lung parenchyma with uniform alveoli and clear bronchiolar
lumen (Fig. 5.1A, B) and there is no hyperplasia and no shedding of cells of the
bronchial epithelium (Fig. 5.1C). Whereas, S group showed emphysematous
changes in the lung parenchyma and bronchi filled with mucus (Fig. 5.1D, E).
High power photomicrograph of bronchial wall from the same section of lung
showed hyperplasia with shedding of cells in the bronchial epithelium (Fig. 5.1F).
Furthermore, S+A group showed a mild, scattered emphysematous change in the
lung parenchyma with few dilated alveoli and mainly clear bronchial lumen with
minimal bronchial secretions (Fig. 5.1G, H). Epithelial hyperplasia is apparent but
no shedding of cells in the bronchial epithelium of this group is observed in high
power photomicrograph (Fig. 5.1I). The liver sections showed no remarkable
difference between the three groups (Fig. 5.2).
5.3.2 Progression of DNA Fragmentation: Comet Assay
Comets with different shapes were obtained from cells of S and S+A, each
shape depicted levels of DNA fragmentation (Fig. 5.3). The background level
of DNA damage was found to be higher in S cells as indicated by significantly
increased tail length, tail extent moments and tail DNA as compared to C
(p<0.05) (Fig. 5.4). The electrophoresis conditions remained fairly constant
over time in all the three groups of cells. The S+A cells although showed
increased tail length, tail extent moments and tail DNA as compared to C cells
but it was decreased as compared to S cells. The percentage increase of tail
length was 23% (p<0.001) in S cells and 11% (p=0.035) in S+A cells as
compared to C, whereas 10% (p=0.04) decrease in S+A cells as compared to S
cells. Similarly, the percentage increase of tail extent moment and tail DNA
was 30% and 14% (p<0.001 and p=0.01, respectively) in S cells and 9% and
5%, respectively in S+A cells as compared to C, whereas 17% (p=0.03) and
8% decrease in S+A cells as compared to S cells. However, the olive tail
moment showed no remarkable difference between the three groups.
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A 100X
D 100X
G 100X
B 200X
E 200X
H 200X
C 400X Control (C)
F 400X Cigarette Smoke treated (S) I 400X Cig. Smoke + Antioxidant (S+A)
Figure 5.1 Histological presentation of lung tissues
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A 100X
B 400X
Control (C)
C 100X
E 100X
D 400X
Cigarette Smoke (S)
F 400X
Cig. Smoke + Antioxidant treated (S+A)
Figure 5.2 Histological presentation of liver tissues
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A.
B.
C.
Figure 5.3 Comet Assay A. Control group, B. Smoke exposed group, C.
Antioxidant prior to smoke exposed group
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Histological, Molecular and Biochemical Study of Mouse Exposed to Acute Cigarette Smoke
140
#
∗∗ ∗
Extent of Length/Moment
120
C
100
S
S+A
80
60
∗∗
#
∗
40
20
t
om
en
NA
il M
il D
Ta
O
il E
xt
liv
e
Ta
Ta
en
Ta
tM
il L
en
om
gt
en
h
t
0
Figure 5.4 Tail length, tail extent moment, tail DNA and olive tail moments of
comets in control (C), smoke (S) and smoke along with antioxidant (S+A)
exposed mice. Values are expressed as a percentage of those obtained in control
group and represent means ± S.D. The intra- and inter-assay coefficients of
variation were less than 10%.
∗∗
p<0.001 vs. control, ∗p<0.05 vs. control, #p<0.05 vs. smoke treated
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Histological, Molecular and Biochemical Study of Mouse Exposed to Acute Cigarette Smoke
5.3.3 Expression Analyses by Reverse Transcription PCR (RT-PCR)
5.3.3.1 CYBA, mEPHX, GSTP1 Gene Expression in Lungs
The CYBA mRNA expression was significantly increased in S (1.25-fold) and
S+A (1.16-fold), versus C group (p=0.001 and p=0.02, respectively) (Fig. 5.5).
Similarly, mEPHX mRNA expressions was significantly increased in S (1.5fold) and S+A (1.22-fold) as compared to C group (p<0.001). Moreover, the
lung of S+A group mice showed 19% decrease in mEPHX expression as
compared to S (p=0.02). However the GSTP1 mRNA expression was
significantly decreased in S (1.16-fold) and S+A (1.1-fold) vs. C group
(p=0.02 and p=0.09, respectively).
5.3.3.2 CYBA, mEPHX, GSTP1 and CYP1A2 Gene Expression in Liver
In the liver the CYBA mRNA expression was significantly decreased in S+A
(1.22-fold) vs. C group (p=0.03) (Fig. 5.6). Moreover, the S+A showed 1.2fold decrease in CYBA expression as compared to S group (p=0.03). Similarly,
mEPHX mRNA expressions was significantly increased in S (1.54-fold) as
compared to C (p<0.001). However, the liver of S+A showed 1.2-fold decrease
in mEPHX expression as compared to S (p=0.017). However the GSTP1 and
CYP1A2 mRNA expression showed no remarkable difference between the
three groups (Fig. 5.6).
5.3.4 Immunoblot Analysis of mEPHX and GSTP1 in Lung and Liver
As shown in Fig. 5.7 acute smoke exposure alone resulted in significant increase
expression of mEPHX in both lungs and liver as compared to controls. However,
S+A group showed decrease in expression of mEPHX in lungs and liver as
compared to S group but increased as compared to C. In lungs there was
significant increase in expression of mEPHX as compared C group.
Expression of GSTP1 did not alter in the three groups either in case of lungs or
liver.
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β-actin
∗∗
180
#
C
∗∗
S
160
% of Control
140
∗∗
∗
S+A
120
∗
100
80
60
40
20
0
CYBA
mEPHX
GSTP1
Figure 5.5 Quantitative analysis of CYBA, mEPHX, GSTP1 and β-actin mRNA
expression in lungs of control (C), smoke (S) and smoke+antioxidant (S+A)
exposed mice. The reverse transcription PCR experiments were repeated thrice and
the images shown are the best representation of the data. The graphical representation
shows the relative integrated densitometry values (IDV) quantified and normalized by
that of β-actin signal using AlphaEaseFC software. Values are expressed as a
percentage of those obtained in control group and represent means ± S.D.
∗∗
p<0.001 vs. control, ∗p<0.05 vs. control, #p<0.05 vs. smoke treated
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Histological, Molecular and Biochemical Study of Mouse Exposed to Acute Cigarette Smoke
β-actin
180
∗∗
C
160
S
#
% of Control
140
120
100
S+A
#
∗
80
60
40
20
0
CYBA
mEPHX
GSTP1
CYP1A2
Figure 5.6 Quantitative analysis of CYBA, mEPHX, GSTP1, CYP1A2 and β-actin
mRNA expression in liver of control (C), smoke (S) and smoke+antioxidant
(S+A) exposed mice. The experiments were repeated thrice and the images shown are
the best representation of the data. The graphical representation shows the relative
integrated densitometry values (IDV) quantified and normalized by that of β-actin
signal using AlphaEaseFC software. Values are expressed as a percentage of those
obtained in control group and represent means ± S.D.
∗∗
p<0.001 vs. control, ∗p<0.05 vs. control, #p<0.05 vs. smoke treated
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Histological, Molecular and Biochemical Study of Mouse Exposed to Acute Cigarette Smoke
160
∗∗
∗
140
∗
Fold Expression
120
100
C
80
S
S+A
60
40
20
0
Lung
Liver
Figure 5.7 Immunoblot and quantitative analysis for mEPHX protein levels in
lung and liver of control (C), smoke (S) and smoke along with antioxidant (S+A)
exposed mice. The immunoblots were repeated thrice and the images shown are the
best representation of the data. The graphical representation shows the relative
integrated densitometry values (IDV) quantified and normalized by that of α-tubulin
(loading control) signal using AlphaEaseFC software. Values are expressed as a
percentage of those obtained in control group and represent means ± S.D.
∗∗
p<0.001 vs. control, ∗p<0.05 vs. control
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Histological, Molecular and Biochemical Study of Mouse Exposed to Acute Cigarette Smoke
5.3.5 Biochemical Parameters in Lung and Liver
As shown in Table 5.2 acute smoke exposure alone resulted in increased activity
of antioxidant enzymes like CAT, GRx and GST and increased levels of GSH and
LPO in lungs as compared to controls. In liver also same trend was observed
except for CAT activity which decreased as compared to C. However, S+A group
showed decrease in the activities of CAT, GRx and GST and GSH, LPO levels in
lungs as compared to S group but increased as compared to C. In the liver also
same trend was observed except for CAT activity and LPO, GSH levels, which
decreased as compared to S and C group both.
The lungs of S group had increased CAT, GRx, GST activities by 2.67, 1.27,
1.63-fold (p<0.001, p<0.001 and p=0.012, respectively) and GSH level by 1.32fold (p=0.032) as compared to C group (Fig. 5.8A). Whereas, S+A group had
increased GRx and GST activities by 1.16-fold and 1.52-fold (p=0.01 and p=0.008,
respectively) as compared to C group. Moreover, the S+A group showed decrease
in the CAT, GRx activities by1.9 fold and 1.1 fold (p=0.001 and p=0.02,
respectively) and LPO levels by 1.1-fold (p=0.01) as compared to S group (Fig.
5.8B). The GPx activity significantly decreased in S and S+A group by 1.16-fold
and 1.18 fold, respectively (p<0.001) as compared to C group.
The liver of S group showed increase in GRx, GST activities by 1.3-fold, 2.32fold (p=0.001 and p<0.001, respectively; Fig. 5.9A) and LPO level by 1.5- fold
(p<0.001, Fig. 5.9B) as compared to C group. However, S+A group showed
increase of GRx and GST activities by 1.22-fold and 1.84-fold (p=0.009 and
p<0.001, respectively) as compared to C group. Whereas, S+A group showed
decrease of GST activity by1.26 fold (p<0.001) and LPO and GSH levels by 1.68fold and 1.28-fold (p<0.001 and p=0.035, respectively) as compared to S group.
The CAT activity decreased in S and S+A group by 2.67-fold and 2.90-fold,
respectively (p<0.001) as compared to C.
In liver, the SOD activity increased in S and S+A group by 1.28-fold and 1.27fold, (p=0.035 and p=0.04, respectively) as compared to C (Figure 5.10).
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Histological, Molecular and Biochemical Study of Mouse Exposed to Acute Cigarette Smoke
Table 5.2 Levels and activities of different antioxidants and oxidative stress markers
Controls
(n=5)
Smoke treated
(n=5)
NAC+smoke
treated (n=5)
*p
**p
***p
LPO (nmole MDA formed/hr/gm of protein)
2.46 ± 0.02
2.50 ± 0.023
2.30 ± 0.082
NS
NS
0.01
GSH (nmole/mg of tissue)
0.70 ± 0.25
0.93 ± 0.15
0.86 ± 0.23
0.032
NS
NS
SOD (U/min/mg of protein)
1.48 ± 0.44
1.21 ± 0.28
1.45 ± 0.38
NS
NS
NS
CAT (nmole of H202 consumed /min/mg of protein)
148.0 ± 88.38
396.0 ± 72.2
206.8 ± 69.1
<0.001
NS
0.001
GPx (nmoles of NADPH oxidized/min/mg protein)
379.4 ± 7.57
326.6 ± 3.03
320.2 ± 12.81
<0.001
<0.001
NS
GRx (nmoles of NADPH oxidized/min/mg protein)
142.7 ± 10.61
182.6 ± 3.75
165.9 ± 12.51
<0.001
0.01
0.02
GST (nmole of CDNB conjugate/min/mg of protein)
90.2 ± 5.28
147.6 ±17.14
137.9 ± 15.76
0.012
0.008
NS
LPO (nmole MDA formed/hr/gm of protein)
2.43 ± 0.06
3.75 ± 0.20
2.23 ± 0.04
<0.001
NS
<0.001
GSH (nmole/mg of tissue)
0.93 ± 0.23
0.95 ± 0.22
0.74 ± 0.26
NS
NS
0.035
SOD (U/min/mg of protein)
1.59 ± 0.39
2.04 ± 0.22
2.03 ± 0.26
0.035
0.04
NS
CAT (nmole of H202 consumed /min/mg of protein)
381.6 ± 56.4
142.8 ± 51.6
131.6 ± 49.97
<0.001
<0.001
NS
GPx (nmoles of NADPH oxidized/min/mg protein)
263.6 ± 32.21
254.0 ± 29.15
234.4 ± 34.56
NS
NS
NS
GRx (nmoles of NADPH oxidized/min/mg protein)
207.9 ± 30.14
270.4 ± 13.54
254.9 ± 16.0
0.001
0.009
NS
GST (nmole of CDNB conjugate/min/mg of protein)
177.6 ±14.36
413.5 ±19.91
328.5 ± 11.58
<0.001
<0.001
<0.001
Lung
Liver
n, number of animals; Values are expressed as means ± SD; *p between Controls and Smoke Treated; **p between controls and NAC+smoke treated;
***p between Smoke Treated and NAC+smoke treated
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Histological, Molecular and Biochemical Study of Mouse Exposed to Acute Cigarette Smoke
A
∗∗
500
450
C
400
∗∗
S
∗∗
S+A
#
300
250
#
Actvities
350
∗∗ ∗
200
∗
150
∗
100
50
0
CAT
GPx
GRx
GST
Lung Biochem icals
B
3
#
2.5
C
S
Levels
2
S+A
1.5
∗
1
0.5
0
LPO
GSH
Lung Biochem icals
Figure 5.8 CAT, GPx, GRx, GST activities enzyme activities (A) and LPO,
GSH levels (B) in lung of control (C), smoke (S) and smoke along with
antioxidant (S+A) exposed mice. Values are expressed as a percentage of those
obtained in control group and represent means ± S.D. The assays were repeated
twice and the intra- and inter-assay coefficients of variation were less than 10%.
∗∗
p<0.001 vs. control, ∗p<0.05 vs. control, #p<0.05 vs. smoke treated
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Histological, Molecular and Biochemical Study of Mouse Exposed to Acute Cigarette Smoke
A
500
∗∗
450
#
∗∗
400
Activities
350
∗∗
300
250
∗∗
200
C
∗
S
∗∗
S+A
150 A.
100
50
A.
0
CAT
GPx
GRx
GST
Liver Biochem icals
B
∗∗
4
3.5
3
#
Levels
2.5
C
2
S
S+A
1.5
#
1
0.5
0
LPO
GSH
Liver Biochem icals
Figure 5.9 CAT, GPx, GRx, GST activities enzyme activities (A) and LPO,
GSH levels (B) in liver of control (C), smoke (S) and smoke along with
antioxidant (S+A) exposed mice. Values are expressed as a percentage of those
obtained in control group and represent means ± S.D. The assays were repeated
twice and the intra- and inter-assay coefficients of variation were less than 10%.
∗∗
p<0.001 vs. control, ∗p<0.05 vs. control, #p<0.05 vs. smoke treated
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Histological, Molecular and Biochemical Study of Mouse Exposed to Acute Cigarette Smoke
2.5
∗
∗
Activity
2
1.5
C
S
1
S+A
0.5
0
Lung
Liver
SOD
Figure 5.10 SOD enzyme activities of in lung and liver of control (C), smoke
(S) and smoke along with antioxidant (S+A) exposed mice. Values are
expressed as a percentage of those obtained in control group and represent means
± S.D. The assays were repeated twice and the intra- and inter-assay coefficients
of variation were less than 10%.
∗
p<0.05 vs. control
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Histological, Molecular and Biochemical Study of Mouse Exposed to Acute Cigarette Smoke
5.4 Discussion
The assessment of COPD risk from exposure to acute term CS must take into
account potential interactions of the toxic chemicals of smoke and its
detoxification. CS could influence the metabolic activation of smoke chemicals by
phase I enzymes, the inactivation of reactive intermediate metabolites by phase II
enzymes and the induction of the enzymes catalyzing these reactions (Vakharia et
al, 2001a,b). In this study, we focused our attention on the possible influence of
acute term CS exposure on the induction of xenobiotic metabolizing enzymes in
lungs and livers of mice.
At first, the histological analysis of lung and liver revealed that the smoke exposed
group showed emphysematous changes in the lung parenchyma with bronchi filled
with mucus, however, the lung of animal treated with antioxidant, NAC, prior to
cigarette smoke exposure showed a mild emphysematous change with few dilated
alveoli and mainly clear bronchial lumen. Moreover the liver did not show any
significant change in acute term exposure. This may be due to the inhalation
exposure of smoke for short period of time, hence shows its effect on direct exposed
organ like lung only. Moreover the oxidants induced by CS can directly damage
components of the lung extracellular matrix such as elastin and collagen or even
modify the matrix to make it more susceptible to protease attack (Rahman I, 2005).
Specific
proteases
derived
from
alveolar
macrophages
(AMs)
and
polymorphonuclear cells (PMNs) are responsible for lung injury (Churg et al, 2002).
Through comet assay we showed that inhaled CS induces DNA SSB in mice;
antioxidant NAC could prevent those effects, suggesting that free radicals in CS
cause damage. High concentrations of free radicals are present in both the gas and
the tar phase of CS and free radicals play an important role in induction of DNA
SSB. It has been shown that the highly reactive hydroxyl radicals are involved in
the formation of DNA SSB in vitro (Nakayama et al, 1985). This DNA SSB is
inhibited by antioxidant enzymes such as catalase and superoxide dismutase and
hydroxyl radical scavengers, sodium benzoate and dimethylthiourea (Leanderson
and Tagesson, 1992; Nakayama et al, 1985). Our results of acute CS and NAC
exposure are in confirmation of these earlier reports.
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Histological, Molecular and Biochemical Study of Mouse Exposed to Acute Cigarette Smoke
Furthermore RT-PCR and immunoblots analyses revealed the expression of
detoxifying and oxidative stress genes in lung and liver of mice. Acute CS
exposure lead to induction of genes, mEPHX and CYBA in lung and liver both.
GSTP1 and CYP1A2 genes also shows induction in liver, however decrease
expression of GSTP1 in lungs. The antioxidant NAC exposure lead to decreased
expression of above genes as compared to S group however still remained
elevated as compared to C group. Conversely, statistically significant induction of
mEPHX protein were observed after exposure to CS in both lung and liver. This is
in agreement with the literature, GST gene induction might rely on the glutathione
conjugation of epoxide-containing B(a)P metabolites, on one hand, and of B(a)Pinduced lipid peroxidation products, on the other hand (Borlak and Thum, 2001;
Sun et al, 1996; Ueng et al, 1998). Accordingly, the induction of mEPHX, CYBA,
GSTP1 and CYP1A2 genes is consistent with the well-characterized mechanism of
induction of target genes by PAHs (Whitlock, 1999).
Moreover at biochemical level activity/levels of several detoxifying and oxidative
stress enzymes were estimated to study the effect of acute CS and NAC exposure.
The activity/levels of most of the biochemical increased by CS, however NAC
reciprocate this. The generation of ROS (O2•−), resulted from smoke exposure, are
the first event in the development of lung oxidative injury (Kinnula VL, 2005).
SOD is the primary enzyme in defending the lung against the damaging effects of
O2•−, which rapidly dismutate O2•− to more stable ROS, H2O2. Both CAT and GPx
catalyze the dismutation of H2O2 to H2O and molecular O2 (Rahman and Adcock,
2006). The decrease in the enzyme activity of SOD, CAT and GPx will lead to
increased oxidative stress. The major enzymatic antioxidants in the airways are
CAT and SOD (Rahman I, 2005). In our study induction of oxidants are much
higher than that of the activity/levels of antioxidants, which suggest the
deleterious effect of oxidants in short term CS exposure.
In conclusion, this is the first report showing the toxicological effect and the
signaling mechanism of acute-term exposure of CS in mice lungs and liver. The
acute study interestingly shows impact of CS mostly in lungs by a mechanism that
CS intermediates should elicit a local action on pulmonary tissue prior to exert
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Histological, Molecular and Biochemical Study of Mouse Exposed to Acute Cigarette Smoke
any systemic action (Bilimoria and Ecobichon, 1992). Importantly, acute smoke
exposure did result in deleterious effect, as observed by increased products of
lipid peroxidation, MDA and decreased antioxidants. The MDA is a stable end
product of lipid peroxidation, which can cause oxidative damage in DNA, lipids
and proteins. The deleterious effect of CS in acute exposure is due to higher
expression and activity/levels of oxidants than that of antioxidants. The delicate
balance that exists between the toxicity of oxidants and the protective effects of
intra and extracellular antioxidant defense systems is critical for the maintenance
of normal function. This study further contributes to the understanding of the
deleterious effects of acute-term exposure of CS in lungs and liver.
147