Long term ethanol consumption leads to lung tissue oxidative stress and injury.
ABSTRACT Alcohol abuse is a systemic disorder. The deleterious health effects of alcohol consumption may result in irreversible organ damage. By contrast, there currently is little evidence for the toxicity of chronic alcohol use on lung tissue. Hence, in this study we investigated long term effects of ethanol in the lung.
Though body weight of rats increased significantly with duration of exposure compared to its initial weight, but there was no significant change in relative weight (g/100 g body weight) of lung due to ethanol exposure. The levels of thiobarbituric acid reactive substances (TBARS), nitrite, protein carbonyl, oxidized glutathione (GSSG), redox ratio (GSSG/GSH) and GST activity elevated; while reduced glutathione (GSH) level and activities of glutathione reductase (GR), glutathione peroxidase (GPx), catalase, superoxide dismutase (SOD) and Na(+)K(+) ATPase reduced significantly with duration of ethanol exposure in the lung homogenate compared to the control group. Total matrix metalloproteinase activity elevated in the lung homogenate with time of ethanol consumption. Histopathologic examination also demonstrated that severity of lung injury enhanced with duration of ethanol exposure.
16-18 weeks old male albino Wistar strain rats weighing 200-220 g were fed with ethanol (1.6 g/ kg body weight/ day) up to 36 weeks. At the end of the experimental period, blood samples were collected from reteroorbital plexus to determine blood alcohol concentration, and the animals were sacrificed. Various oxidative stress related biochemical parameters, total matrix metalloproteinase activity and histopathologic examinations of the lung tissues were performed.
Results of this study indicate that long term ethanol administration aggravates systemic and local oxidative stress, which may be associated with lung tissue injury.
- SourceAvailable from: sciencedirect.com[Show abstract] [Hide abstract]
ABSTRACT: Chronic alcohol abuse is a systemic disorder and a risk factor for acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). A significant amount of ingested alcohol reaches airway passages in the lungs and can be metabolized via oxidative and non-oxidative pathways. About 90% of the ingested alcohol is metabolized via hepatic alcohol dehydrogenase (ADH)-catalyzed oxidative pathway. Alcohol can also be metabolized by cytochrome P450 2E1 (CYP2E1), particularly during chronic alcohol abuse. Both the oxidative pathways, however, are associated with oxidative stress due to the formation of acetaldehyde and/or reactive oxygen species (ROS). Alcohol ingestion is also known to cause endoplasmic reticulum (ER) stress, which can be mediated by oxidative and/or non-oxidative metabolites of ethanol. An acute as well as chronic alcohol ingestions impair protective antioxidants, oxidize reduced glutathione (GSH, cellular antioxidant against ROS and oxidative stress), and suppress innate and adaptive immunity in the lungs. Oxidative stress and suppressed immunity in the lungs of chronic alcohol abusers collectively are considered to be major risk factors for infection and development of pneumonia, and such diseases as ARDS and COPD. Prior human and experimental studies attempted to identify common mechanisms by which alcohol abuse directly causes toxicity to alveolar epithelium and respiratory tract, particularly lungs. In this review, the metabolic basis of lung injury, oxidative and ER stress and immunosuppression in experimental models and alcoholic patients, as well as potential immunomodulatory therapeutic strategies for improving host defenses against alcohol-induced pulmonary infections are discussed.Toxicology Letters 07/2013; · 3.15 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Similar to effects of alcohol on the heart, liver, and brain, the effects of ethanol (EtOH) on lung injury are preventable. Unlike other vital organ systems, however, the lethal effects of alcohol on the lung are underappreciated, perhaps because there are no signs of overt pulmonary disorder until a secondary insult, such as a bacterial infection or injury, occurs in the lung. This paper provides overview of the complex changes in the alveolar environment known to occur following both chronic and acute alcohol exposures. Contemporary animal and cell culture models for alcohol-induced lung dysfunction are discussed, with emphasis on the effect of alcohol on transepithelial transport processes, namely, epithelial sodium channel activity (ENaC). The cascading effect of tissue and phagocytic Nadph oxidase (Nox) may be triggered by ethanol exposure, and as such, alcohol ingestion and exposure lead to a prooxidative environment; thus impacting alveolar macrophage (AM) function and oxidative stress. A better understanding of how alcohol changes the landscape of the alveolar epithelium can lead to improvements in treating acute respiratory distress syndrome (ARDS) for which hospitalized alcoholics are at an increased risk.BioMed research international. 01/2013; 2013:470217.
Oxidative Medicine and Cellular Longevity 3:6, 414-420; November/December 2010; © 2010 Landes Bioscience
Long-term ethanol consumption leads
to lung tissue oxidative stress and injury
414 Oxidative Medicine and Cellular Longevity Volume 3 Issue 6
*Correspondence to: Subir Kumar Das; Email: firstname.lastname@example.org
Submitted: 10/29/10; Revised: 11/25/10; Accepted: 12/06/10
Previously published online: www.landesbioscience.com/journals/oximed/article/14417
The deleterious health effects of alcohol consumption may result
in irreversible organ damage.1 By contrast; the ravages of alcohol
abuse have been viewed as relatively sparing the lung. More than
two centuries ago, Benjamin Rush, the first Surgeon General of
the United States, noted that pneumonia and tuberculosis were
infectious complications more commonly encountered in people
who drank alcohol, and a century later, alcohol abuse as the major
risk factor for pneumonia was cited.2 This risk has largely been
attributed to alterations in immune function and/or structural/
functional defects in the upper airway. In fact, until relatively
recently it had been generally assumed that chronic alcohol abuse
had no effect on the lung itself.3 However, one epidemiologi-
cal finding revealed that alcohol abuse independently increased
the risk for developing the acute respiratory distress syndrome
Background: alcohol abuse is a systemic disorder. The deleterious health effects of alcohol consumption may result in
irreversible organ damage. By contrast, there currently is little evidence for the toxicity of chronic alcohol use on lung
tissue. hence, in this study we investigated long-term effects of ethanol in the lung.
Results: Though body weight of rats increased significantly with duration of exposure compared to its initial weight,
there was no significant change in relative weight (g/100 g body weight) of lung due to ethanol exposure. The levels of
thiobarbituric acid reactive substances (TBaRs), nitrite, protein carbonyl, oxidized glutathione (GssG), redox ratio (GssG/
Gsh) and GsT activity elevated; while reduced glutathione (Gsh) level and activities of glutathione reductase (GR), glu-
tathione peroxidase (Gpx), catalase, superoxide dismutase (sOD) and Na+K+aTpase reduced significantly with duration
of ethanol exposure in the lung homogenate compared to the control group. Total matrix metalloproteinase activity
elevated in the lung homogenate with time of ethanol consumption. histopathologic examination also demonstrated
that severity of lung injury enhanced with duration of ethanol exposure.
Methods: 16–18 week-old male albino Wistar strain rats weighing 200–220 g were fed with ethanol (1.6 g/kg body
weight/day) up to 36 weeks. at the end of the experimental period, blood samples were collected from reteroorbital
plexus to determine blood alcohol concentration and the animals were sacrificed. Various oxidative stress-related bio-
chemical parameters, total matrix metalloproteinase activity and histopathologic examinations of the lung tissues were
Conclusions: Results of this study indicate that long-term ethanol administration aggravates systemic and local oxida-
tive stress, which may be associated with lung tissue injury.
subir Kumar Das1,* and sukhes Mukherjee2
1Department of Biochemistry; esI-pGIMsR; Joka, Kolkata; 2amrita Institute of Medical sciences; Cochin, Kerala India
Key words: ethanol, glutathione, lung, oxidative stress, reactive oxygen species
Abbreviations: ARDS, acute respiratory distress syndrome; ECM, extracellular matrix; GSH, reduced glutathione; GSSG, oxidized
glutathione; GPx, glutathione peroxidase; GR, glutathione reductase; GST, glutathione s-transferase; LPO, lipid peroxidation;
MMP, matrix metalloproteinase; ROS, ractive oxygen species; SOD, superoxide dismutase; TBA, thiobarbituric acid; TBARS,
thiobarbituric acid reactive substances; TCA, trichloroacetic acid
(ARDS),4 a devastating form of acute lung injury in which the
air spaces become flooded with inflammatory cells and debris,
leading to respiratory failure and may cause death.5
Ethanol, being soluble both in water and lipids, can diffuse
rapidly through the mucous membrane of the esophagous and
stomach. After its absorption ethanol appears in both expired air
and in urine. It is not stored in the body, as whatever is ingested
is oxidized.6,7 When consumed in moderate amounts, the major
part of the ethanol is metabolized primarily in the liver by alco-
hol dehydrogenase,8 a cytosolic enzyme with multiple isoforms.9
Alcohol can also be metabolized in microsomes via the cyto-
chrome P-450 component CYP2E1.10 This enzyme complex has
a lower affinity for alcohol than the hepatic alcohol dehydroge-
nase enzyme and therefore may not contribute significantly to
overall alcohol metabolism following occasional use.9 Alcohol is
metabolized in the lung through the cytochrome P-450 system.11
www.landesbioscience.com Oxidative Medicine and Cellular Longevity 415
(SOD) activities after 12 weeks in the lung homogenate (Table 3).
However, GST activity increased significantly after 12 weeks of
ethanol exposure compared to the control (0 weeks) or 4 weeks of
ethanol exposed groups (Table 3). Moreover, there were significant
differences in glutathione peroxidase (GPx), glutathione s-transfer-
ase (GST) and catalase activities between 12 weeks and 24 weeks
of ethanol exposure (Table 3). Interestingly, no significant change
in these oxidative stress related parameters was observed between
24 weeks and 36 weeks of ethanol exposed lung homogenates of
rats (Tables 2 and 3).
Total matrix metalloproteinase (MMP) activity increased sig-
nificantly in ethanol exposed lung tissues. The activity showed a
progressive increase, attaining maximum on 24th week after etha-
nol exposure (Fig. 1A and B). Histopathological analysis showed
that the broncholar and normal alveolar structure was preserved
in the control specimen (Fig. 2A), wheras degenerative alveolar
structures and leukocytic infiltration were observed in the lung
tissues of the ethanol exposed groups (Fig. 2B–E). Severity
of inflammation increased with duration of ethanol exposure
(Fig. 2B–E). In the controls, median scores of leukocyte infiltra-
tion were mainly under 0 and 1, whereas this score and severity of
infiltration increased with duration of ethanol exposure (Table 4).
The intragastric ethanol infusion technique allowed maximal
ethanol consumption and absolute control over ethanol-induced
In addition, during alcohol ingestion, alcohol freely diffuses
from the bronchial circulation directly through the ciliated epithe-
lium where it vaporizes as it moves into the conducting airways.
Moreover, vaporized alcohol can deposit back into the airway lin-
ing fluid to be released again into the airways during exhalation.
This “recycling” of alcohol vapor results in repeated exposure of
the airway epithelium to high local concentrations of alcohol.12
Therefore, we investigated long-term effects of ethanol in the
lung in this study.
Body weight of ethanol exposed rats increased significantly after
12 weeks compared to the control group (0 week) and continued
to increase (Table 1). However, there was no significant change
in relative weight (g/100 g body weight) of lung with duration
of ethanol exposure (Table 1). Once exposed to ethanol, plasma
alcohol levels remain unchanged in rats (Table 1).
Ethanol exposure significantly increased nitrite and protein
carbonyl content after 4 weeks; thiobarbituric acid reactive sub-
stances (TBARS) level, oxidized glutathione (GSSG) content
and redox ratio (GSSG/GSH) after 12 weeks; while significantly
decreased reduced glutathione (GSH) level after 12 weeks in com-
parison to the control group in the lung homogenate (Table 2).
Compared to control group, ethanol exposure significantly
reduced GPx and Na+K+ATPase activities after 4 weeks, while
glutathione reductase (GR), catalase and superoxide dismutase
Table 1. Body weight, relative weight of lung and plasma ethanol profile of ethanol treated rats for different time period
4 week 12 week24 week36 weekF varianceSignificance
Nil1.6 g/kg 1.6 g/kg1.6 g/kg1.6 g/kg--
Body weight (g) 210.0 ± 7.07225.0 ± 11.83255.83 ± 10.68a,b
265.8 ± 11.14a,b
284.17 ± 10.7a,b,c
Relative weight of lung
(g/100 g body weight)
0.65 ± 0.020.62 ± 0.020.64 ± 0.010.65 ± 0.02 0.62 ± 0.022.334 0.083
plasma alcohol level (mM)- 54.83 ± 3.0653.33 ± 2.7352.0 ± 2.3652.67 ± 1.96647.142<0.001
Values are mean ± sD of 6 rats in each group. p values: a<0.001 compared to control group (Group 1); b<0.001 compared to 4 week ethanol treated
group (Group 2); c<0.01 compared to 12 week ethanol treated group (Group 3). No significant change was observed between 24 weeks and 36 weeks
of ethanol exposed rats.
Table 2. effect of ethanol on thiobarbituric acid reactive substances (TBaRs), nitrite, protein-carbonyl, reduced glutathione (Gsh) and oxidized
glutathione (GssG) levels and on redox ratios (GssG/Gsh) of lung homogenate
Control (0 week)0.51 ± 0.0213.46 ± 0.760.97 ± 0.137.00 ± 0.090.58 ± 0.010.08 ± 0.001
4 week 0.71 ± 0.0720.33 ± 2.66a
1.6 ± 0.14a
6.68 ± 0.12 0.60 ± 0.020.09 ± 0.003
12 week 0.86 ± 0.16b
24.48 ± 2.45a,e
1.88 ± 0.22a
4.12 ± 0.21a,d
0.62 ± 0.01c
0.15 ± 0.005a,d
24 week0.97 ± 0.17a,f
28.8 ± 1.57a,d,h
2.21 ± 0.19a,d,i
3.83 ± 0.31a,d
0.69 ± 0.02a,d,g
0.18 ± 0.012a,d,i
36 week1.0 ± 0.17a,e
31.26 ± 1.3a,d,g
2.33 ± 0.17a,d,h
3.66 ± 0.33a,d,i
0.71 ± 0.03a,d,g
0.19 ± 0.019a,d,i
Values are mean ± sD of 6 rats in each group. p values: a<0.001, b<0.01, c<0.05 compared to control group (Group 1); d<0.001, e<0.01, f<0.05 compared to
4 week ethanol treated group (Group 2); g<0.001, h<0.01, i<0.05 compared to 12 week ethanol treated group (Group 3). No significant change was ob-
served between 24 weeks and 36 weeks of ethanol exposed rats *, μmole MDa formed/min/100 mg tissue; #, nmole/g wet tissue; ƒ, nmole/mg protein;
Ψ, μg/mg tissue.
416 Oxidative Medicine and Cellular Longevity Volume 3 Issue 6
antioxidants results in oxidative stress, and may cause cellular
damage by peroxidation of membrane lipids, sulfhydryl enzyme
inactivation, protein cross-linking and DNA breakdown. This
damage may be involved in the etiology of diverse human dis-
eases.16,18-22 Consequently, organ damage may further increase
One of the proposed mechanisms of chronic ethanol induced-
toxicity is the membrane damage due to the direct effect of lipid
peroxidation products,13 i.e., TBARS, which was found to be
increased in the ethanol exposed rats in the present study (Table 2).
Chronic alcohol ingestion enhanced superoxide generation in the
lung tissue.23 Protein nitration has been suggested to be a final
product of highly reactive nitrogen oxide intermediates (e.g., per-
oxynitrite) formed in reactions between NO and oxygen-derived
species such as superoxide. Nitrite is a stable metabolite of NO in
vivo. Increased nitrite level was observed in lung homogenate of
ethanol exposed rats in the present study (Table 2) through the
organ injury. In the present study, a dose of ethanol 1.6 g/kg body
weight/day was used based on our previous observation,13 where
we found that this amount of ethanol exposure was tolerable
for long period, causing organ damage and is partially reverse-
bile during abstaining.14 However, continuous ethanol exposure
maintained plasma ethanol level in a steady state in our study
(Table 1) is in agreement with other report.15 It is also assumed
that alcohol metabolism through the cytochrome p-450 system
in the lung is significant11 and may be sufficient to exert signifi-
cant oxidative stress in the lung,16,17 due to their unique structure
Reactive oxygen species (ROS) are constantly produced in
the cells, but under normal physiological conditions the enzy-
matic and non-enzymatic antioxidant mechanisms of the cell
overcome the destructive potential of ROS. There is a delicate
balance between the production of ROS and endogenous pro-
tection mechanisms. Overproduction of ROS or a decrease in
Table 3. effect of ethanol on glutathione reductase (GR), glutathione peroxidase (Gpx), glutathione s-transferase (GsT), catalase, superoxide
dismutase (sOD) and Na+K+-aTpase activities in lung homogenate
Control (0 week) 14.95 ± 1.2624.82 ± 0.714.43 ± 0.083.26 ± 0.08 1.36 ± 0.08223 ± 9.55
4 week13.38 ± 1.07 19.57 ± 1.24a
4.5 ± 0.093.1 ± 0.09 1.28 ± 0.09171 ± 10.41a
12 week11.35 ± 0.89a,e
15.67 ± 1.5a,d
6.58 ± 0.26ad
2.91 ± 0.11a,f
1.08 ± 0.11a,f
134.3 ± 7.2a,d
24 week10.12 ± 0.77a,d
13.15 ± 0.79a,d,i
7.13 ± 0.28a,d,h
2.65 ± 0.1a,d,h
0.97 ± 0.1a,d
124 ± 8a,d
36 week9.07 ± 1.04a,d,h
11.67 ± 1.42a,d,g
7.5 ± 0.37a,d,g
2.58 ± 0.11a,d,g
0.93 ± 0.08a,d
118.5 ± 12.48a,d
Fvariance41.465113.506212.244 47.75223.292 120.875
Values are mean ± sD of 6 rats in each group. p values: a<0.001, b<0.01, c<0.05 compared to control group (Group 1); d<0.001, e<0.01, f<0.05 compared
to 4 week ethanol treated group (Group 2); g<0.001, h<0.01, i<0.05 compared to 12 week ethanol treated group (Group 3). No significant change was
observed between 24 weeks and 36 weeks of ethanol exposed rats. *, nmole NaDph breakdown/min/mg protein; #, μmole CDNB conjugate formed/
min/mg protein; ƒ, μmole h2O2decomposed/min/mg protein; Ψ, One unit of the enzyme was the amount of sOD capable of inhibiting by 50% the rate
of NaDh oxidation observed in the control. The specific activity was expressed as units/mg protein; ω, nmole pi/mg/protein/h.
Figure 1. Changes in the total activity of matrix metalloproteinases in ethanol exposed rat lung with time. Rats were fed with ethanol (1.6 g/kg body
weight/day). (a) extracts of lung tissue samples (100 μl) from control and ethanol exposed rats of different time interval (up to 36 weeks) were copoly-
merised with acrylamide-bisacryamide containing gelatin in Tris buffer (ph 8.8). after polymerization, the gels were then incubated in substrate buf-
fer, stained with Coomasie brilliant blue and destained with methanol-acetic acid-water. a blank was prepared without enzyme. (B) activity measured
by densitometric analysis. Values given are average of 3 experiments ±sD.
www.landesbioscience.com Oxidative Medicine and Cellular Longevity 417
and so almost all intracellular glutathione is reduced. During an
oxidative stress, there will be a flux of glutathione to the oxidized
form, and the ratio of oxidized to reduced glutathione may then
be indication of this stress.27 Decreased GSH level, increased
GSSG level and redox ratio with duration of ethanol exposure
indicated time dependent elevation of oxidative stress in the lung
in this study (Table 2).
Chronic ethanol ingestion resulted in significant decrease in
GPx activity in the lung may be due to either free radical depen-
dent inactivation of enzyme or depletion of its substrates i.e.,
GSH and NADPH.13,14 Glutathione s-transferase (GST) plays
an essential role by eliminating toxic compounds by conjugating
them with glutathione. Increased GST activity, and decreased
GPx and GR activities (Table 3), followed by thiol depletion
(Table 2) are important factors in sustaining a pathogenic role
for oxidative stress.13,14,28
The presence of superoxide dismutase (SOD) in various com-
partments of human body enables SOD to dismutate superoxide
radical immediately.13 The cytochrome P450 2E1 was demon-
strated to generate higher amounts of H2O2,29 and is linked to
increased generation of hydroxyl radicals.30 Decreased SOD and
catalase activities with duration of ethanol exposure in the lung
in this study (Table 3) may be due to loss of NADPH, or genera-
tion of superoxide, or increased lipid peroxidation or combina-
tion of all.13
activation of a constitutive nitric oxide synthase (NOS), most
likely the endothelial NOS isoform (eNOS or NOS-3).24
The tissue GSH concentration reflects its potential for detoxi-
fication and is critical in preserving the proper cellular redox
balance for its role as a cellular protectant.25 During the detoxifi-
cation of lipid and other peroxides produced by free radical attack,
glutathione peroxidase converts glutathione from a reduced state
(GSH) to an oxidized one (GSSG).26 The NADPH-dependent
enzyme glutathione reductase converts GSSG back to GSH,
Figure 2. Lung tissues fixed in formalin, processed for hematoxylin and eosin stain to assess morphological changes under microscope. (a) Normal
texture of lung in control animals; (B) 4 weeks ethanol treated lung with mild inflammation; (C) 12 weeks ethanol treated rat-Lung alveoli with marked
interstitial infammation with inflammatory exudates in some of the alveoli; (D) 24 weeks ethanol treated rats-Lung alveoli filled with inflammatory
exudates; (e) 36 weeks ethanol treated rat-severe inflammation.
Table 4. Leokocyte infiltration score of the control and ethanol
GroupLeukocyte infiltration score
Leukocyte infiltration was evaluated to determine the severity of oxida-
tive damage in the lung. each section was divided into subsections,
and leukocyte infiltration was assessed using the scale for comparison
at a magnification of X400: 0 = no extravascular leukocytes; 1 ≤ 10
leukocytes; 2 = 10 - 45 leukocytes; and 3 ≥ 45 leukocytes.
418 Oxidative Medicine and Cellular Longevity Volume 3 Issue 6
for 24 wks; Group 5: ethanol (1.6 g ethanol/kg body wt/day)
treated for 36 wks.
At the end of the experimental period, blood samples were
collected from retro-orbital plexus of rats. The animals were
then sacrificed after overnight fast, by intraperitoneal injection
of sodium pentobarbital (50 mg/kg body wt). The lung was dis-
sected out, cleaned with ice-cold saline, blotted dry and imme-
diately transferred either to the ice chamber for biochemical
studies or fixed in 10% buffered formal saline for histopathologi-
cal examinations. Various oxidative stress related non-enzymes
such as TBARS, nitrite, protein carbonyl, GSH, GSSG; and
enzymes activities such as GR, GPx, GST, catalase, SOD and
Na+K+ATPase were estimated. The Animal Ethics Committee of
the Institution approved the procedures in accordance with the
Biochemical methods. Blood collected from retro-orbital
plexus was used for blood ethanol concentration estimation using
an ethanol assay kit (Sigma).
Lung samples were homogenized in 0.25 M sucrose solution,
and were used to estimate tissue protein.39
Determination of thiobarbituric acid reactive substances
(TBARS). Lung samples were homogenized in ice-cold 0.25 M
tris buffer (pH 7.4). 0.3 ml of this homogenate was mixed thor-
oughly with 2 ml of TCA-TBA-HCl [trichloroacetic acid (TCA)
15% w/v, thiobarbituric acid (TBA) 0.375% w/v and hydrochlo-
ric acid (HCl) 0.25 N]. The solutions were heated for 15 min
in a boiling water bath, cooled; the flocculent precipitates were
removed, and the absorbance was recorded at 535 nm. The extent
of lipid peroxidation was calculated using molar extinction coef-
ficient 1.56 x 105 M-1cm-1.40
Nitrite estimation. Sulfanilamide (1%, 50 μl) in 2.5% ortho-
phosphoric acid (Griess reagent 1), followed by N-(1-naphthyl)
ethylenediamine (0.1%, 50 μl) in distilled water (Griess reagent 2)
were added to the tissue homogenate, incubated in dark at room
temperature for 10 min. The absorbance was measured at 540 nm.
The concentration of nitrite was measured by using NaNO2 as a
Protein-carbonyl content. Proteins were precipitated with
20% trichloroacetic acid and centrifuged. The precipitates were
resuspended in 2,4-dinitrophenylhydrazine (10 mM) and vor-
texed at 10 min intervals for 1 h at room temperature. The pellets
were washed thrice with ethanol/ethyl acetate, resuspended in
0.6 ml of 6 M guanidine hydrochloride, incubated at 37°C for
15 min and centrifuged at 5,000 g for 3 min. The absorbance of
supernatant was measured at 366 nm for carbonyl content, and
calculations were performed with ε value of 22,000 M-1cm-1.42
Glutathione content. The lung (~100 mg) samples were homog-
enized in ice-cold 0.1 M phosphate buffer (pH 7.4). For reduced
glutathione (GSH) content, the homogenates were immediately
mixed with sulfosalicylic acid, shaked well and centrifuged. Each
supernatant fraction was mixed separately with 5,5'-dithiobis(2-
nitrobenzoic acid) (in 0.01 M phosphate buffer, pH 8) and absor-
bance was recorded at 412 nm.43 For oxidized glutathione, 200 μl
supernatant was added to 3.78 ml of water to which 40 μl of
2-vinylpyridine was mixed to mask the GSH and left at room tem-
perature for 3 h before estimation as described above.44
Na+-K+ ATPase participates in lung fluid clearance by creating
the active transport of sodium.31 Oxidative stress plays a role in
mediating the ethanol-induced downregulation of lung Na+-K+
ATPase in this study. GSH depletion seems to be a major deter-
minant of this effect.32 Increased lipid peroxidation combined
with decreased tissue Na+-K+ ATPase activity in the lung in this
study (Table 3) may be associated with impairment of membrane
phospholipids.33 The decreased enzyme activity gives rise to the
disintegration of the cells and consequently to the thickening of
the air-blood barrier, alveolar degeneration and leukocyte infil-
tration,31 as observed histopathologically in the lungs of the eth-
anol-exposed rats (Fig. 2 and Table 4). Ethanol-induced reactive
species causes phenotypic alterations in the lung and alters the
lung’s response to inflammatory stimuli.36 These inflammatory
mediators lead to leukocyte activation, expression of endothelial
adhesion molecules and vascular endothelial damage.34
The alveolar extracellular matrix (ECM) is considered to be a
static structural component of the lung tissue. It serves as a mod-
ulator of cell growth and development, inflammation, angio-
genesis, cell migration, tissue differentiation and repair. There is
growing evidence that aberrant remodeling of the ECM contrib-
utes both to the early inflammatory phase as well as to the later
fibroproliferative phase of the syndrome.35,36 Evidence suggests
that degradation of the ECM by (MMPs) may contribute to the
development of lung injury,37 as observed in this study (Fig. 1).
Though biochemical alterations and oxidative stress related
parameters respond early in alcoholism than the histopathologi-
cal changes;38 leukocyte infiltration and matrix metalloprotein-
ases activation were observed in ethanol-treated lungs and its
severity increased with duration of ethanol exposure in the pres-
ent study. In conclusion, our results suggest that long-term etha-
nol administration aggravates systemic and local oxidative stress,
which is one of the leading causes of lung injury.
Materials. Ethanol was purchased from Bengal Chemicals,
Kolkata. Chemicals from Sisco Research Laboratory (SRL),
India, Sigma Chemical Co., St. Louis, MO; and E. Merck were
used. Ethanol was diluted with distilled water to get desired con-
centration and fed orally, when necessary.
Animals and treatment. The male albino rats (16–18 weeks
old) of Wistar strain weighing 200–220 g were housed in plastic
cages inside a well-ventilated room, with the room temperature
maintained at 25 ± 2°C, with a 12 h light/dark cycle. Animals
had free access of standard diet14 containing (%) bengal gram,
31; gingelly oil cake, 30; wheat, 28; polished rice, 10; salt mix-
ture, 0.5; vitamin-mineral mixture, 0.3; and yeast with fish or
liver oil, 0.2. Food and water were given ad libitum. Animals
were weighed daily and their general condition and behavior were
recorded, including their daily intake of food.
The rats were divided into the following five groups of 6 ani-
mals each. Group 1: control—were fed normal diet and water;
Group 2: ethanol (1.6 g ethanol/kg body wt/day) treated for 4
wks; Group3: ethanol (1.6 g ethanol/kg body wt/day) treated for
12 wks; Group 4: ethanol (1.6 g ethanol/kg body wt/day) treated
www.landesbioscience.com Oxidative Medicine and Cellular Longevity 419
Na+-K+ ATPase activity. Specific activity of Na+-K+ ATPase
estimation is based on the principle that the inorganic phosphate
is released from protein in presence of 3 mM disodium adenosine
5'-triphosphate in incubation medium.49
Multiwell zymogram (Total MMP activity). 100 μl tissue
samples were placed in 24-well containing plate and incubated at
37°C for 30 mins for enzyme activation. Zymo gel (15 mg gela-
tin dissolved in 3.75 ml of Tris buffer pH (8.8), 3.75 ml acryl-
amide-bisacryamide (30:0.3), 7.125 ml double distilled water,
150 μl 10% ammonium persulphate (freshly prepared) and 15
μl TEMED) was added and allowed to settle for 1 h. The gels
were then placed in 6-well containing plates with zymo buffer
(calcium chloride buffer, pH 7.5: 3.03 g Tris-HCl and 0.36 mg
CaCl2 dissolved in 500 ml double distilled water) and incubated
overnight. After removing the zymo buffer, the gels were stained
with Coomasie brilliant blue for 3 to 4 h. The gels were finally
destained with methanol-acetic acid-water.50
Histopathological examination. Lung tissues fixed in 10%
formalin, routinely processed and embedded in paraffin. Sections
were cut (4 μm thick) and stained with hematoxylin and eosin
to assess morphological changes under microscope. Leukocyte
infiltration was evaluated to determine the severity of oxidative
damage that resulted from ethanol intoxication. Each section
was divided into 10 subsections, and leukocyte infiltration was
assessed using the following scale for comparison at a magnifica-
tion of x400: 0 = no extravascular leukocytes; 1 ≤ 10 leukocytes;
2 = 10–45 leukocytes; and 3 ≥ 45 leukocytes.
Statistical analysis. Results were expressed as mean ± SD
(standard deviation). All statistical analysis were performed by
one-way analysis of variance (ANOVA) with multiple compari-
son tests and student’s t-test using the Statistical Package for
Social Sciences, version 11 (SPSS, Chicago, IL). The values of
significance were evaluated with p values. The differences were
considered significant at p < 0.05.
Glutathione reductase (GR, EC 184.108.40.206) activity. The tis-
sues were homogenized in phosphate buffer (0.12 M, pH 7.2),
and were mixed to 15 mM EDTA in phosphate buffer and
9.6 mM NADPH. The reaction was initiated by adding
oxidized glutathione (GSSG, 65.3 mM). Change in absor-
bance was monitored at 340 nm; and the specific activity
was determined using extinction coefficient for NADPH of
Glutathione peroxidase (GPx, EC 220.127.116.11) activity.
Glutathione peroxidase activity was measured based on the
principle that oxidized glutathione produced by GPx is reduced
at a constant rate by glutathione reductase with NADPH as
a cofactor. NADPH allows maintaining predictable levels of
reduced glutathione. The oxidative rate of NADPH was moni-
tored at 340 nm.46
Glutathione-s-transferase (GST; EC 18.104.22.168) activity. The
tissues were homogenized using phosphate buffer (0.05 M, pH
6.5). 1-chloro-2,4-dinitrobenzene (CDNB) in phosphate buf-
fer was mixed with reduced glutathione, and then tissue extract
was added. The change in absorbance was monitored at 340
nm, and calculated from extinction coefficient 9.6 mM-1cm-1.47
Catalase (EC 22.214.171.124) activity. The tissues were homog-
enized in 0.05 M phosphate buffered saline (pH 7.0). The rate
of decomposition of H2O2 (2 μl, 30%) in 0.05 M phosphate
buffer (1 ml, pH 7.0) at 240 nm after addition of homogenized
tissue was noted. The specific activity was calculated assuming
molar extinction coefficient 22,000 M-1cm-1 at 240 nm.14
Superoxide dismutase (SOD, EC 126.96.36.199) activity.
Superoxide dismutase activity was measured by the inhibition
of auto-oxidation of 0.2 mM pyrogallol (air equilibriated) in 50
mM at Tris-HCl buffer (pH 8.2) containing 1 mM diethylene-
triamine pentaacetic acid. The rate of autooxidation was moni-
tored at 420 nm. The inhibition of pyrogallol autooxidation
was initiated by addition of tissue homogenate.48
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