Tissue and Cell 43 (2011) 384391

Contents lists available at SciVerse ScienceDirect

Tissue and Cell
j our nal home page: www. el sevi er . com/ l ocat e/ t i ce
Chronic ethanol consumption induces histopathological changes and increases
nitric oxide generation in the rat liver
Luis F. Tirapelli
a
, Marcelo E. Batalho
b
, Ana L. Jacob-Ferreira
a
, Daniela P. Tirapelli
a
, Evelin C. Carnio
b
,
Jos E. Tanus-Santos
a
, Regina H. Queiroz
c
, Sergio A. Uyemura
c
, Cludia M. Padovan
d
, Carlos R. Tirapelli
b,
a
Faculty of Medicine of Ribeiro Preto, University of So Paulo (USP), Brazil
b
College of Nursing of Ribeiro Preto, USP, Brazil
c
Faculty of Pharmaceutical Sciences of Ribeiro Preto, USP, Brazil
d
Faculty of Philosophy Science and Letters of Ribeiro Preto, USP, Brazil
a r t i c l e i n f o
Article history:
Received 22 March 2011
Received in revised form11 August 2011
Accepted 17 August 2011
Available online 17 September 2011
Keywords:
Ethanol
Liver
Nitric oxide
Metalloproteinase
Histopathological changes
a b s t r a c t
In the present work we evaluated the effect of ethanol consumption in histopathological liver changes
and several biochemical biomarkers employed in the detection of hepatic dysfunction. Male Wistar
rats were treated with ethanol 20% (vol/vol) for 6 weeks. Histopathological investigation of livers from
ethanol-treated animals revealed steatosis. Indices of hepatic function (transaminases) and mitochon-
drial respiration were not altered in ethanol-treated rats. Chronic ethanol consumption did not alter
malondialdehyde (MDA) levels in the liver. Ethanol consumption induced a signicant increase on hepatic
nitrite and nitrate levels. Treatment with ethanol increased both mRNA expression and immunostain-
ing of iNOS, but not eNOS. Finally, ethanol consumption did not alter hepatic levels of metalloproteinase
(MMP)-2 and MMP-9. We conclude that alterations on biochemical biomarkers (nitrite and nitrate levels)
and histopathology occurred in ethanol-treated rats, supporting the practice of including both types of
evaluation in toxicity studies to detect potential ethanol-related hepatic effects. In our model of ethanol
consumption, histopathological liver changes were accompanied by elevation in nitrite and nitrate levels
indicating increased nitric oxide (NO) generation. Since iNOS-derived NO contributes to hepatic injury,
the increased levels of NO described in our study might contribute to a progressive hepatic damage.
Therefore, increases in NO generation may be an early indicator of ethanol-induced liver damage.
2011 Elsevier Ltd. All rights reserved.
1. Introduction
Alcoholic liver disease is one of the major causes of morbidity
and mortality among alcoholics in the world (Grant et al., 1988). It
is proposed that excessive consumption of ethanol causes liver dis-
ease by a combination of several pathologic mechanisms including
oxidative stress, inammation, redox alterations, and mitochon-
drial damage (Beier and McClain, 2010). For this reason a wide
variety of new biomarkers reecting liver status are being used to
evaluate the hepatic effect of excessive ethanol consumption.
The impact of ethanol consumption in hepatic function is
traditionally evaluated by histological analysis and measure-
ments of serum transaminases (Conigrave et al., 2003). Ethanol

Corresponding author at: Departamento de EnfermagemPsiquitrica e Cincias

Humanas, Laboratrio de Farmacologia, Universidade de So Paulo, Escola de Enfer-
magem de Ribeiro Preto, Avenida Bandeirantes 3900, 14040-902, Ribeiro Preto,
SP, Brazil. Tel.: +55 16 36020532.
E-mail address: crtirapelli@eerp.usp.br (C.R. Tirapelli).
induces oxidative stress in the liver (Dey and Cederbaum, 2006),
which is associated with enhanced production of reactive oxygen
species (ROS). The latter damage lipids and proteins, resulting in
increased malondialdehyde (MDA) levels, which have been used as
a biomarker for ethanol-induced hepatic dysfunction (Baldi et al.,
1993). In addition to ROS, nitric oxide (NO) is also important in the
development of hepatic injury. NO is produced froml-arginine by
the catalic action of NOsynthase (NOS) (Moncada and Higgs, 1993),
whichis anenzymethat occurs inthreemajor isoforms: endothelial
(eNOS), neuronal (nNOS) and inducible (iNOS) (Forstermann et al.,
1998). iNOS fromKupffer cell play a role in alcoholic liver disease,
since ethanol toxicity was signicantly attenuated in iNOS knock-
out miceandinmicetreatedwith1400W, aselectiveiNOSinhibitor
(McKim et al., 2003). In fact, sustained generation of NO by iNOS,
may turn on a broad spectrumof sequelae, fromlipid peroxidation
to DNA damage and pro-apoptotic effects (Davis et al., 2001) impli-
cating iNOS to the pathogenesis of ethanol-induced liver injury. On
the other hand, NO produced from eNOS may be protective, since
inhibition of this enzyme enhanced liver damage (McKim et al.,
2003).
0040-8166/$ see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tice.2011.08.003
L.F. Tirapelli et al. / Tissue and Cell 43 (2011) 384391 385
Metalloproteinases (MMPs) are a family of zinc-dependent
proteinases that are involved in the pathophysiology of many
hepatic diseases (Consolo et al., 2009). Penumathsa et al. (2006)
observed that rats treated with ethanol presented liver damage
and increased MMPs activities. More recently, Koken et al. (2010)
demonstrated that ethanol consumption induces hepatic dysfunc-
tion and increases plasma levels of MMP-9, implicating MMPs as
potential biochemical indicators of ethanol-induced hepatic dys-
function.
The histopathological progression of alcoholic liver disease is
well-characterized and is actually a spectrum of liver diseases,
ranging from steatosis, to inammation and necrosis (steatohep-
atitis), to brosis and cirrhosis (Mann et al., 2003). However, liver
biopsypresents somelimitations suchas theriskof adiseaseunder-
estimation, as hepatic lesions are often irregularly located within
the liver (Sorensen et al., 2003). Moreover, in some cases, alter-
ation of biochemical biomarkers precedes the histopathological
liver changes (Travlos et al., 1996). In this line, early detection of
altered biochemical biomarkers could contribute to early diagnosis
of ethanol-induced hepatic liver disease before histopathological
changes develop. For this reason, in the present study we aimed
to simultaneously analyze the effect of ethanol consumption on
biochemical biomarkers and histopathological liver changes.
2. Materials and methods
2.1. Experimental design
Therats werehousedunder standardlaboratoryconditions with
free access to food and water. The housing conditions and experi-
mental protocols were approved by the Animal Ethics Committee
of the University of So Paulo Campus of Ribeiro Preto. Male
Wistar rats, initially weighing 250280g (5070 days old) were
randomly divided into two groups: control and ethanol. Control
rats received water ad libitum; and rats from the ethanol group
received 20% (vol/vol) ethanol in their drinking water (Tirapelli et
al., 2008a). The ethanol-treated group was submitted to a brief and
gradual period adaptation: the animals received 5% ethanol in their
drinking water in the rst week, 10% in the second week and 20% in
the third week (all values in volume ratios). At the end of the third
weektheexperimental stagebegan. Therats werethentreatedfor 6
weeks andthelivers wereremovedfor biochemical andhistological
analysis.
2.2. Blood ethanol and serummeasurements of glucose, aspartate
aminotransferase (AST), alanine aminotransferase (ALT), alkaline
phosphatase, total proteins and bilirubin
Bloodwas collectedfromthe inferior vena cava of anaesthetized
rats (thiopental sodium, 0.4mg/kg, i.p.) using heparinzed syringes,
and the samples were placed in 10-mL headspace vials by adding
1.0g sodiumchloride, 1.0mL water, 100L of the internal standard
(acetonitrile) solution and 1.0mL of blood. Ethanol analysis was
carriedout using a CG-17Agas chromatographer (Shimadzu, Kyoto,
Japan) equipped with a ame-ionization detector and an HSS-4A
headspace sampler (Shimadzu). Calibrations standards were pre-
pared in the same headspace vials (0.103.16mg/mL). The results
are expressed as mg ethanol per mL blood (Tirapelli et al., 2008a).
For glucose, AST, ALT, alkaline phosphatase, total proteins and
bilirubin measurements, blood was collected from the inferior
vena cava of anaesthetized rats. The samples were centrifuged
at 800010,000g for 10min at room temperature. The serum
was assayed using commercial kits (Labtest Diagnstica, So Paulo,
SP, Brazil) with the auto-analyzer ABBOTT (model ABAA VP). The
results were expressed as units per litre, grams per litre or mil-
ligrams per litre (Tirapelli et al., 2008a).
2.3. Histopathological evaluation
For histology, tissue samples xed in 10% buffered formalin
were parafnembeddedfor preparationof 5msections that were
stained with Massons trichrome. The photomicrographs were
taken using a binocular Zeiss

microscope (model Axioskop 2 plus)

with magnication 100 and 400.
2.4. Hepatic mitochondrial function
Liver mitochondria were prepared in 0.25mol/L sucrose
1mmol/L ethylenediamine tetraacetic acid at pH7.2 and 4

C using
standard centrifugation procedures. Mitochondrial protein and
mitochondrial respiratory function were measured as previously
described (Tirapelli et al., 2008b). Briey, oxygen consumption was
determined at 30

C with a Clark oxygen electrode (Gilson Med-

ical Electronics, Middleton, Wis, USA) in a thermostat-controlled
chamber. Mitochondria (2mg) were added to 1.5mL of solution
containing (in mmol/L): 125.0 sucrose, 65.0 KCl, 10.0 Hepes, 1.0
MgCl
2
, 2.0 KH
2
PO
4
, 0.1 EGTA, and 2mol/L rotenone, adjusting
the pH to 7.4 with KOH. Mitochondrial respiration was initi-
ated by addition of succinate (5mmol/L nal concentration), and
oxidative phosphorylation by addition of 200mmol/L ADP. Oxy-
gen consumption recordings allowed the calculation of V3 [rate of
state 3 (ADP-stimulated) respiration], of V4 [rate of state 4 (non-
ADP-stimulated) respiration], and of the respiratory control ratio
(RCR=V3/V4). The oxygen uptake of V3 and V4 was expressed
in nmol oxygen/min/mg mitochondrial protein. Mitochondrial
swelling was determined in a hypotonic buffer by measuring the
decrease in the absorbance at 540nm, using a Beckman DU-640
spectrophotometer.
2.5. Assessment of lipid peroxide levels in the liver
Lipid peroxide levels in the rat liver were determined by mea-
suring thiobarbituric acid reactive substances using a uorometric
method as previously described (Yagi, 1998). This method requires
excitation at 515nm and emission at 553nm and uses 1,1,3,3-
tetramethoxypropane as standard. The lipoperoxide levels were
expressed in terms of malondialdehyde (MDA, nmol/mL). MDA
results were normalized for protein concentration assessed with
the Bradford technique.
2.6. Levels of nitrite and nitrate
Nitrite and nitrate levels were measured in supernatants from
total liver homogenates prepared under liquid N
2
as previously
described (Lizarte et al., 2009). Aliquots of 5L were injected into a
Sievers chemiluminescence analyzer (model 280) and pelleted by
centrifugation with VCl
3
and HCl (at 95

C), which act as reductants

for nitrate, or NaI and acetic acid, which function as reductants for
nitrite. Results were normalized for protein concentration assessed
with the Bradford technique and are expressed as mol/L/mg pro-
tein.
To evaluate plasma nitrate levels, blood was collected into
chilled plastic tubes, containing heparin (200U), centrifuged for
20min at 2,000g at 4

C for plasma separation and stored at 70

C
before dosage. On the day of the assay, plasma samples were
thawed and deproteinized with 95% ethanol (at 4

C) for 30min,
subsequently centrifuged, and the supernatant was used for mea-
surement of nitrate as described above. Results are expressed as
mol/L.
386 L.F. Tirapelli et al. / Tissue and Cell 43 (2011) 384391
2.7. Quantitative real-time polymerase chain reaction (qRT-PCR)
for eNOS and iNOS
Total cellular RNA was extracted using Trizol

Reagent (Invit-
rogen, Carlsbad, CA, USA), and RNA was reverse-transcribed to
single-stranded cDNA using a High Capacity Kit (Applied Biossys-
tems, Foster City, CA, USA) according to manufacturers protocol.
For the quantitative analysis of the genes of interest, which con-
sisted of eNOS (Rn02132634 s1) and iNOS (Rn00561646 m1),
we used the commercially available TaqMan Assay-on-Demand
System, which consists of oligonucleotides and probes (Applied
Biosystems, Foster City, CA, USA). Reverse transcription was per-
formed using 1g total RNA for each sample in 20L of the total
reaction mixture. The cDNA obtained was diluted 1:10, and 4.5L
was used for each 10L of the RQ-PCR mixture using the TaqMan
Master Mix (Applied Biosystems). All reactions were carried out
in duplicate and analyzed with the 7500 Sequence Detection Sys-
temapparatus (Applied Biosystems). Data were analyzed using the
ABI-7500 SDS software. The total RNA absorbed was normalized
on the basis of the Ct value for the GAPDHgene (Rn 01775763 m1).
The variation in expression among samples was calculated by the
2
Ct
method, with the mean delta Ct value for a group of 6
samples fromcontrol rats used for calibration (Lizarte et al., 2009).
2.8. Immunohistochemistry
Parafn embedded liver segments were stained by the avidin-
biotinylated peroxidase complex (ABC) method. Briey, 4m
sections (Reichert Jung 2040 microtome) were cut and went
through a deparafnization protocol with xylene and ethanol.
Endogenous peroxydase and biotin were blocked by immersing
slides in 3% hydrogen peroxide. The following primary antibod-
ies were incubated: iNOS (1126-1144; N7782, SigmaAldrich,
Missouri, USA) diluted 1:200 and eNOS (1185-1205; N3893,
SigmaAldrich, Missouri, USA) diluted 1:200. The reactions were
revealed using 0.2mg/mL diaminobenzidine solution (10mg
tablets in 50mL PBS 0.01M pH 7.4; D5905; SigmaAldrich, Mis-
souri, USA) and stained by Harris hematoxylin. In each slide, two
elds were selected in areas with the higher concentration of posi-
tive cells, or stained cells using 400 magnication. Positive and
negative stained cells were counted. Results were expressed as
percent of positive cells. The slides were analyzed using Leica
microscope, model DM 5500 B. The images were registered by a
Leica digital camera DFC 290 (3MP) attached in the microscope,
and led by Leica QWin software.
2.9. Measurement of hepatic MMP-2 and MMP-9 activities by
gelatin zymography
Tissues were homogenized in buffer containing 20mmol/L
TrisHCl (pH 7.4), 1mmol/L 1,10-phenanthroline, 1mmol/L PMSF,
1mmol/L NEM, and 10mmol/L CaCl
2
. Briey, tissue extracts were
subjected to electrophoresis on 7% SDS-PAGE gels co-polymerized
with gelatin (1%) as the substrate. After electrophoresis, the gels
were incubated for 1h at room temperature in a 2% Triton X-100
solution, washed two times with water and incubated at 37

C for
16h in TrisHCl buffer (pH 7.4), containing 10mmol/L CaCl
2
. The
gels were xed with 30% methanol and 10% acetic acid, stained
with 0.05% Coomassie Brilliant Blue G-250, and then destained
with 30% methanol and 10% acetic acid. Evidence of gelatinolytic
activity was distinguished by the presence of unstained bands
against the background of the Coomassie blue-stained gelatin. The
staining differences were assayed by densitometry using a Kodak
Electrophoresis Documentation and Analysis System (EDAS) 290
(Kodak, Rochester, NY). Intergel analysis was possible after the nor-
malization of gelatinolytic activity with an internal standard (fetal
Table 1
Blood ethanol levels and serum levels of glucose, AST, ALT, total proteins, alkaline
phosphatase, total bilirubin and direct bilirubin obtained fromcontrol and ethanol-
treated rats.
Control Ethanol
Blood ethanol (mg/mL) ND (6) 1.60.2 (15)
Glucose (mg/dL) 97.27.2 (18) 101.46.8 (15)
AST (U/L) 107.57.1 (16) 118.25.1 (14)
ALT (U/L) 52.13.0 (16) 56.33.5 (18)
Total proteins (g/L) 68.30.8 (18) 68.70.7 (18)
Alkaline phosphatase (U/L) 172.213.1 (15) 163.37.5 (16)
Total bilirubin (mg/L) 3.80.1 (16) 3.40.3 (17)
Direct bilirubin (mg/L) 1.70.2 (17) 1.60.1 (16)
AST, aspartate aminotransferase; ALT, alanine aminotransferase; ND, non-
detectable. Numbers betweenparentheses indicate the number of replicates. Values
shown are meanSEM.
bovine serum2%). The MMP-2 and MMP-9 were identiedas bands
at 72 and 92kDa, respectively, which were inhibited by phenan-
throline and not by other proteinase inhibitors, and were further
identiedbyimmunoprecipitationwithspecic antibodies. Results
are reported as ratio between bands density and internal standard
value. Drugs and reagents were purchased fromSigmaAldrich (St.
Louis, MO).
2.10. Statistical analysis
Data are presented as meanstandard error of the mean (SEM).
Statistically signicant differences were calculated by Students t
test. p<0.05 was considered as statistically signicant.
3. Results
3.1. Body mass
The body weights of the rats prior to treatment averaged
2715g (n=25) in the control group and 2687g (n=23) in the
ethanol group. Animals receiving ethanol in the drinking water for
6 weeks showed reduced body weight (45512g) in comparison
to age-matched control rats (51811g) (p<0.05; Students t-test).
3.2. Blood ethanol and serummeasurements of glucose, AST, ALT,
alkaline phosphatase, total proteins and bilirubin
Results for bloodethanol levels andserumlevels of glucose, AST,
ALT, alkaline phosphatase, total proteins and bilirubin are summa-
rized in Table 1. Ethanol levels were increased in animals given
ethanol in drinking water (approximately 34mmol/L). The values
of the hepatic biomarkers did not differ between the groups.
3.3. Histopathological evaluation
No histopathological alterationwas observedinlivers fromcon-
trol rats (Fig. 1A). The presence of some areas of interstitial edema
parallel to the cords of hepatocytes (Fig. 1B) was observed in the
liver parenchyma from ethanol-treated rats. In liver parenchyma
from ethanol-treated rats was observed the presence of hepa-
tocytes with cytoplasmic vacuoles scattered (Fig. 1C) and the
presence of macrogoticular diffuse steatosis showing diffuse bul-
lous hepatocytes (Fig. 1D).
3.4. Hepatic mitochondrial function
No differences were found in mitochondrial respiration
between livers fromcontrol or ethanol-treated rats (Table 2).
L.F. Tirapelli et al. / Tissue and Cell 43 (2011) 384391 387
Fig. 1. (A) Photomicrograph of the liver parenchyma fromcontrol animals. Hepatic venule (v); cords of hepatocytes (*); 400. (B) Overviewshowing the presence of cords of
hepatocytes adjacent to areas of interstitial edema (*) around three hepatic venules (V) in an animal in the group with ethanol, 100. (C) Detail at 400showing the presence
of hepatocytes with cytoplasmic vacuoles (arrows). (D) Presence of macrogoticular diffuse steatosis in the liver parenchyma showing diffuse bullous hepatocytes (arrows);
400. Massons trichrome.
Table 2
Hepatic mitochondrial function in control and ethanol-treated rats.
Control Ethanol
State 3 respiration (nmol O
2
/min/mg protein) 52.2 2.8 48.0 9.5
State 4 respiration (nmol O
2
/min/mg protein) 12.2 1.1 14.1 2.4
Respiration control ratio (state 3/state 4) 4.2 0.5 3.5 0.2
Membrane swelling (absorbance) 1.5 0.12 1.7 0.11
Values are meanSEMof 5 animals for each group.
3.5. Lipid peroxide levels in the liver
NodifferenceontheMDA levels was foundbetweentissues from
control and ethanol-treated rats (Fig. 2).
Fig. 2. Effect of ethanol consumptiononMDA levels inthe rat liver. The experiments
for MDA levels were performed in the liver from control or ethanol-treated rats.
The results were normalized for protein concentration assessed with the Bradford
technique. Values are meanSEMof 6 animals for each group.
3.6. Levels of nitrite and nitrate
Fig. 3 shows that ethanol consumption induced a signi-
cant increase in hepatic nitrite and nitrate levels. Plasma nitrate
levels decreased in ethanol-treated animals (38.11.2mol/L,
n=5) when compared to control animals (54.33.7mol/L, n=5)
(p<0.05; Students t-test).
3.7. eNOS and iNOS mRNA levels
The results obtained by qRT-PCR show that the treatment with
ethanol increased the hepatic mRNA levels (fold 2
Ct
) of iNOS.
On the other hand, no differences in the mRNA expression of eNOS
were detected between tissues from control and ethanol-treated
rats (Fig. 4).
3.8. Immunohistochemistry for eNOS and iNOS
Immunohistochemical positive staining for the NOS isoforms
(iNOS and eNOS) were found by focal distributed throughout the
liver parenchyma without any association with the portal space or
the central hepatic venule (Fig. 5A). Ethanol consumptionincreased
the hepatic staining of iNOS. No differences were found in hepatic
staining of eNOS between the groups (Fig. 5B).
3.9. Hepatic MMP-2 and MMP-9 activity
Fig. 6A shows a representative zymogram of hepatic extracts
showing MMP-2 and MMP-9 bands. Livers from ethanol-treated
rats showed no differences in the activities of both MMP-2 and
MMP-9 compared with those fromcontrol animals (Fig. 6B).
388 L.F. Tirapelli et al. / Tissue and Cell 43 (2011) 384391
Fig. 3. Effect of ethanol consumption on nitrite and nitrate levels in the rat liver.
The experiments for nitrite and nitrate generation were performed in the liver from
control or ethanol-treated rats. The results were normalized for protein concentra-
tion assessed with the Bradford technique. Values are meanSEMof 6 animals for
each group.*Compared to control group (p<0.05; Students t test).
4. Discussion
Moderate ethanol consumption is generally considered to be in
the range 13 drinks/day (Klatsky et al., 1992; Thun et al., 1997),
giving rise to blood ethanol levels of approximately 525mmol/L.
In alcoholics, blood ethanol levels can reach 100mmol/L (Kalant,
1971). Thus, the concentrations of ethanol found in our study
(34mmol/L) are physiologically relevant. In the present study,
histopathological investigation of livers fromethanol-treated ani-
mals revealed that the only observed pathological change was
steatosis, which is one of the earliest hepatic changes caused by
ethanol (Day and James, 1998). In fact, the progression of alcoholic
liver disease is well-characterized and is actually a spectrum of
liver diseases, ranging fromsteatosis, to inammation and necrosis
(steatohepatitis), to brosis and cirrhosis, and eventually hepa-
tocellular carcinoma in some cases. It is important to note that
the risk of alcoholic liver disease increases in a dose- and time-
dependent manner with consumption of ethanol (Mann et al.,
2003). Moreover, environmental (e.g., cigarette smoking, obesity)
or genetic factors (e.g., gender polymorphisms in key genes) might
contribute to overall risk (Day, 2000). Although ethanol consump-
tion induces histopathological changes, we found that indices of
hepatic function (transaminases, bilirubin, alkaline phosphatase
and total proteins) were not altered in serumfromethanol-treated
rats. Thus, a point that might account for the lack of effect of ethanol
on hepatic function is the period of treatment employed in our
study.
Mitochondria are a target for ethanol intoxication. Morpho-
logical and functional changes in mitochondria are one of the key
hallmarks of chronic ethanol exposure in both alcoholics and in
experimental models (Dey and Cederbaum, 2006). In our study no
differences in mitochondrial respiration or swelling were found.
The present data suggests that the effect of ethanol in the rat liver
could be in an initial phase and that exposure of these animals
Fig. 4. Effect of ethanol consumption on eNOS and iNOS mRNA expression in the
rat liver. The mRNA levels of eNOS (top) and iNOS (bottom) were quantied by
qRT-PCR. The results are presented as the expression of the individual mRNAs with
normalization to the housekeeping gene GAPDH by using the Ct method. Values
are meanSEM of 6 animals for each group.*Compared to control group (p<0.05;
Students t test).
to a longer period of ethanol treatment would aggravate the
histopathological alterations here observed and induced hepatic
and mitochondrial dysfunction.
The liver is the mainorganresponsible for metabolizing ethanol.
Themajor routeof ethanol metabolismis theoxidationof ethanol to
acetaldehyde. Rat liver contains alcohol dehydrogenase, anenzyme
that oxidizes ethanol to generate acetaldehyde (Beier and McClain,
2010). The oxidation of acetaldehyde by the acetaldehyde dehy-
drogenase generates ROS that are able to induce cell membranes
damage (Lieber, 1988). The accumulation of ROS leads to damage
in cellular components such as lipids and proteins, resulting in an
increase in MDA levels. Our ndings showno alterations in the lev-
els of MDA in livers from ethanol-treated rats, further suggesting
that ethanol consumption did not induce lipid peroxidation in our
experimental model.
Ethanol is widely distributedinbody water invivo. It has a broad
range of toxicological actions, some of which overlap with those
thought to be modulated or mediated by NO (Deng and Deitrich,
2007). Measurement of tissue nitrite and nitrate provides a reliable
andquantitativeestimateof NOformationinvivo, afreeradical that
is also important in the development of hepatic injury (Baraona et
al., 2002; Deng and Deitrich, 2007). Our data corroborates previous
ndings describing that ethanol consumption increases NO levels
in the rat liver (Baraona et al., 2002) and other tissues such as
vascular endothelial cells (Venkov et al., 1999) and aorta from
female rats (Tirapelli et al., 2008c). On the other hand, nitrate
plasma levels were decreased in ethanol-treated rats, corroborat-
ing previous ndings in the literature (Husain et al., 2004, 2005).
The observation that ethanol increased nitrate generation in the
L.F. Tirapelli et al. / Tissue and Cell 43 (2011) 384391 389
Fig. 5. Representative immunohistochemical photomicrographs (top panels) of positive staining (arrows) for eNOS antibody (A: control group; B: ethanol group) and iNOS (C:
control group; D: ethanol group) in the liver parenchyma, showing labeling in the cytoplasmof hepatocytes. Hepatic venule (hv); 100. Bar graphs represents the percentage
of positive stained cells for eNOS and iNOS in the liver fromcontrol or ethanol-treated rats. Values are meanSEMof 5 animals for each group.*Compared to control group
(p <0.05; Students t test).
rat liver and decrease nitrate levels in the plasma suggests that the
effect of ethanol consumption on NO generation is tissue-specic.
Involvement of NO in the pathogenesis of inammation and
some degenerative disorders has been linked to the generation of
peroxynitrite (ONOO

) (Beier and McClain, 2010). NO can interact

with superoxide anion (O
2

) to form ONOO

, which is a power-
ful oxidant, causing a number of potentially dangerous reactions
including lipid peroxidation, DNA cleavage and reduced vasodilat-
ing activity (Ischiropoulos et al., 1992). MDA, frequently used to
showthe involvement of free radicals in cell damage, is one of the
nal products of lipid peroxidation. However, as mentioned before
no alterations in MDA levels in the liver fromethanol-treated rats
were found, suggesting that ethanol consumption did not induce
lipid peroxidation.
The mRNA expression of iNOS, but not eNOS was increased
by ethanol consumption. Moreover, immunohistochemical
assays showed increased immunostaining for iNOS, indicating
that ethanol consumption up-regulates iNOS expression at the
pre-translational level. Nevertheless, the mechanisms by which
ethanol up-regulates iNOS expression and whether it involves
direct or indirect pathways are not yet clear. One possible expla-
nation may involve the ethanol-induced enhancement in plasma
levels of endotoxin. In this line, El-Mas et al. (2008) showed that
endotoxemia up-regulates the cardiac expressionof iNOS infemale
rats treated with ethanol. In fact, endotoxemia is associated with
activation of iNOS (Forstermann et al., 1998) and it is described to
play a role in the biological effects of ethanol (Kono et al., 2000; Yin
et al., 2000). Moreover, in response to LPS, young rats exposed to
ethanol in uterus showed increased splenic and ileac iNOS expres-
sion and activity (Gottesfeld et al., 1998; Weisbrodt et al., 1999).
Finally, El-Mas et al. (2011) have recently reported that chronic
ethanol consumption induced a 4-fold increases in plasma endo-
toxin along with signicantly higher myocardial iNOS expression.
In the present study, ethanol consumption did not alter eNOS
expression. In fact, ethanol exhibits a different effect on iNOS and
eNOS expression in a variety of cells and tissues. For example,
390 L.F. Tirapelli et al. / Tissue and Cell 43 (2011) 384391
Fig. 6. (A) Arepresentative SDSPAGEgelatinzymogramof hepatic samples. Molec-
ular weights of MMP-2 and MMP-9 bands were identied after electrophoresis on
7% SDSPAGE. Std: internal standard, C: control, E: ethanol. (B) Values for MMP-2
andMMP-9activities. MMP-2andMMP-9were identiedas bands at 72and92kDa,
respectively. Values are meanSEMof 6 animals for each group.
increased expression of eNOS associated to ethanol consumption
was also described in the cavernosal smooth muscle (Lizarte et al.,
2009) and myocardium (El-Mas et al., 2011). In cultured vascular
endothelial cells ethanol increases the expression of eNOS and the
production of NO (Venkov et al., 1999). A reason for such differ-
ences in ethanol-induced expression of eNOS and iNOS in different
tissues remains unclear. However, dose andlengthof ethanol expo-
sure are suggested to be the main factors affecting ethanols effects
on NO production, NOS activity or NOS expression. Cell type also
contributes to this effect (Deng and Deitrich, 2007).
The increase in hepatic NO bioavailability triggered by iNOS
induction has been fundamentally linked to hepatic dysfunction
(McKim et al., 2003). Sustained, high-output generation of NO by
iNOS, induces lipid peroxidation (Davis et al., 2001) and hepatic
injury (McKimet al., 2003). Thus, the increased production of hep-
atic NO here described could account for the future development
of hepatic dysfunction. However, it is not possible to conclude from
our data whether the increased levels of hepatic NO are solely
maintained by iNOS. Further studies are necessary to elucidate this
point.
MMPs are of signicant biomedical interest because they have
been implicated in many pathologic processes characterized by
dysregulated turnover of connective tissue matrices, suchas occurs
in hepatic disease (Consolo et al., 2009). In some hepatic diseases
the extracellular matrix undergoes a process of remodeling, lead-
ing to new collagen formation and deposition. The major role in
matrix degradation is played by MMPs. For this reason, the concept
of monitoring MMPs levels has gained importance in the diagnosis
of liver diseases (Consoloet al., 2009). Inthepresent study, although
ethanol consumption induced steatosis, no alterations in hepatic
MMP-2 and MMP-9 activities were found.
In summary, we found that alterations of biochemical biomark-
ers (nitrite and nitrate levels) and histopathology occurred in livers
fromethanol-treatedrats, supporting the practice of including both
types of evaluation in toxicity studies to detect potential ethanol-
related hepatic effects. In our model of ethanol feeding, early
histopathological liver change (steatosis) was detected and this
response was accompanied by elevation in NO generation. Since
iNOS-derived NO contributes to hepatic injury, the increased lev-
els of NO described in our study might contribute to a progressive
hepatic damage. Therefore, increases in NO generation may be an
early indicator of liver damage.
Acknowledgements
We thank Sonia Dreossi and Antonio Zanardo Filho for excel-
lent technical support. This study was supported by Fundac o de
Amparo Pesquisa do Estado de So Paulo - FAPESP (Process num-
ber: 06/60076-7).
References
Baldi, E., Burra, P., Plebani, M., Salvagnini, M., 1993. Serum malondialdehyde and
mitochondrial aspartate aminotransferase activity as markers of chronic alcohol
intake and alcoholic liver disease. Ital. J. Gastroenterol. 25 (8), 429432.
Baraona, E., Zeballos, G., Sachet, L., Mar, K.M., Lieber, C.S., 2002. Ethanol consump-
tion increases nitric oxide production in rats, and its peroxynitrite-mediated
toxicity is attenuated by polyenylphosphatidylcholine. Alcohol Clin. Exp. Res.
26, 883889.
Beier, J.I., McClain, C.J., 2010. Mechanisms andcell signalinginalcoholic liver disease.
Biol. Chem. 391, 12491264.
Conigrave, K.M., Davies, P., Haber, P., Whiteld, J.B., 2003. Traditional markers of
excessive alcohol use. Addiction 98 (Suppl. 2), 3143.
Consolo, M., Amoroso, A., Spandidos, D.A., Mazzarino, M.C., 2009. Matrixmetallopro-
teinases and their inhibitors as marlers of inammation and brosis in chronic
liver disease. Int. J. Mol. Med. 24, 143152.
Davis, K., Martin, E., Turko, I., Murad, F., 2001. Novel effects of NO. Annu. Rev. Phar-
macol. Toxicol. 41, 203236.
Day, C.P., James, O.E., 1998. Hepatic steatosis: innocent bystander or guilty party?
Hepatology 27, 14631466.
Day, C.P., 2000. Who gets alcoholic liver disease: nature or nurture? J. R. Coll. Phys.
Lond. 34, 557562.
Deng, X.S., Deitrich, R.A., 2007. Ethanol metabolismand effects: nitric oxide and its
interaction. Curr. Clin. Pharmacol. 2 (2), 145153.
Dey, A., Cederbaum, A.I., 2006. Alcohol and oxidative liver injury. Hepatology 43
(Suppl.), S63S74.
El-Mas, M.M., Fan, M., Abdel-Rahman, A.A., 2008. Endotoxemia-mediatedfacilitation
of cardiac iNOS expression accounts for the hypotensive effect of ethanol in
female rats. J. Pharmacol. Exp. Ther. 324, 368375.
El-Mas, M.M., Fan, M., Abdel-Rahman, A.A., 2011. Upregulation of cardiac NOS due
to endotoxemia and vagal overactivity contributes to the hypotensive effect of
chronic ethanol in female rats. Eur. J. Pharmacol. 650 (1), 317323.
Forstermann, U., Boissel, J.P., Kleinert, H., 1998. Expressional control of the con-
stitutive isoforms of nitric oxide synthase (NOS I and NOS III). FASEB J. 12,
773790.
Gottesfeld, Z., Maier, M., Mailman, D., Lai, M., Weisbrodt, N.W., 1998. Splenic sym-
pathetic response to endotoxin is blunted in the fetal alcohol-exposed rat: role
of nitric oxide. Alcohol 16, 1924.
Grant, B.F., Dufour, M.C., Harford, T.C., 1988. Epidemiology of alcoholic liver disease.
Semin. Liver Dis. 8, 1225.
Husain, K., Mejia, J., Lalla, J., Kazim, S., 2004. Time response of alcohol-induced alter-
ations in blood pressure, nitric oxide and oxidant to antioxidant balance in the
plasma of rats. Exp. Clin. Cardiol. 9 (4), 229234.
Husain, K., Mejia, J., Lalla, J., Kazim, S., 2005. Dose response of alcohol-induced
changes in BP, nitric oxide and antioxidants in rat plasma. Pharmacol. Res. 51
(4), 337343.
Ischiropoulos, H., Zhu, L., Beckman, J.S., 1992. Peroxynitrite formation from
macrophage-derived nitric oxide production. Arch. Biochem. Biophys. 298,
446451.
Kalant, H., 1971. Effects of ethanol on the nervous system. In: Tremolleres, J. (Ed.),
International Encyclopedia of Pharmacology and Therapeutics: Alcohols and
Derivatives. Pergamon, NewYork, pp. 189236.
Klatsky, A.L., Armstrong, M.A., Friedman, G.D., 1992. Alcohol and mortality. Ann. Int.
Med. 117, 646654.
Koken, T., Gursoy, F., Kahraman, A., 2010. Long-termalcohol consumption increases
pro-matrix metalloproteinase-9 levels via oxidative stress. J. Med. Toxicol. 6 (2),
126130.
Kono, H., Wheeler, M.D., Rusyn, I., Lin, M., Seabra, V., Rivera, C.A., Bradford, B.U.,
Forman, D.T., Thurman, R.G., 2000. Gender differences in early alcohol-induced
liver injury: role of CD14, NF-B, and TNF. Am. J. Physiol. Gastrointest. Liver.
Physiol. 278, G625G661.
Lieber, C.S., 1988. Biochemical and molecular basis of alcohol-induced injury to liver
and other tissues. N. Engl. J. Med. 319, 16391650.
Lizarte, F.S., Claudino, M.A., Tirapelli, C.R., Morgueti, M., Tirapelli, D.P., Batalho, M.E.,
Carnio, E.C., Queiroz, R.H., Evora, P.R., Tucci Jr., S., Cologna, A., Antunes, E., Mar-
tins, A.C., Tirapelli, L.F., 2009. Chronic ethanol consumption induces cavernosal
smooth muscle dysfunction in rats. Urology 74 (6), 12501256.
Mann, R.E., Smart, R.G., Govoni, R., 2003. The epidemiology of alcoholic liver disease.
Alcohol Res. Health 27 (3), 209219.
L.F. Tirapelli et al. / Tissue and Cell 43 (2011) 384391 391
McKim, S.E., Gabele, E., Isayama, F., Lambert, J.C., Tucker, L.M., Wheeler, M.D., Connor,
H.D., Mason, R.P., Doll, M.A., Hein, D.W., Arteel, G.E., 2003. Inducible nitric oxide
synthase is requiredinalcohol-inducedliver injury: studies withknockout mice.
Gastroenterology 125, 18341844.
Moncada, S., Higgs, A., 1993. The l-arginine-nitric oxide pathway. N. Engl. J. Med.
329, 20022012.
Penumathsa, S.V., Kode, A., Rajagopalan, R., Menon, V.P., 2006. Changes in activities
of MMP in alcohol and thermally oxidized sunower oil-induced liver damage:
NAC antioxidant therapy. Toxicol. Mech. Methods 16 (5), 267274.
Sorensen, H.T., Thulstrup, A.M., Mellemkjar, L., Jepsen, P., Christensen, E., Olsen, J.H.,
Vilstrup, H., 2003. Long-term survival and cause-specic mortality in patients
with cirrhosis of the liver: a nationwide cohort study in Denmark. J. Clin. Epi-
demiol. 56, 8893.
Thun, M.J., Peto, R., Lopez, A.D., Monaco, J.H., Henley, S.J., Heath Jr., C.W., Doll, R.,
1997. Alcohol consumption and mortality among middle-aged and elderly U.S.
adults. N. Engl. J. Med. 337, 17051714.
Tirapelli, C.R., Fukada, S.Y., Yogi, A., Chignalia, A.Z., Tostes, R.C., Bonaventura, D.,
Lanchote, V.L., Cunha, F.Q., de Oliveira, A.M., 2008c. Gender-specic vascular
effects elicited by chronic ethanol consumption in rats: a role for inducible nitric
oxide synthase. Br. J. Pharmacol. 153 (3), 468479.
Tirapelli, C.R., Legros, E., Brochu, I., Honor, J.C., Lanchote, V.L., Uyemura, S.A., de
Oliveira, A.M., DOrlans-Juste, P., 2008a. Chronic ethanol intake modulates
vascular levels of endothelin-1 receptor and enhances the pressor response
to endothelin-1 in anaesthetized rats. Br. J. Pharmacol. 154 (5), 971
981.
Tirapelli, L.F., Bagnato, V.S., Tirapelli, D.P., Kurachi, C., Barione, D.F., Tucci Jr., S.,
Suaid, H.J., Cologna, A.J., Martins, A.C., 2008b. Renal ischemia in rats: mito-
chondria function and laser autouorescence. Transplant. Proc. 40 (5), 1679
1684.
Travlos, G.S., Morris, R.W., Elwell, M.R., Duke, A., Rosenblum, S., Thompson, M.B.,
1996. Frequency and relationships of clinical chemistry and liver and kidney
histopathology ndings in 13-week toxicity studies in rats. Toxicology 107 (1),
1729.
Venkov, C.D., Myers, P.R., Tanner, M.A., Su, M., Vaughan, D.E., 1999. Ethanol increases
endothelial nitric oxide production through modulation of nitric oxide synthase
expression. Thromb. Haemost. 81, 638642.
Weisbrodt, N.W., Lai, M., Gottesfeld, Z., 1999. Ileal nitric oxide formation is altered in
the young rat in response to endotoxin: effects of exposure to alcohol in utero.
Alcohol 17, 247251.
Yagi, K., 1998. Simple assay for the level of total lipid peroxides in serumor plasma.
Methods Mol. Biol. 108, 101106.
Yin, K., Ikejima, M.D., Wheeler, B.U., Bradford, B.U., Seabra, V., Forman, D.T., Sato, N.,
Thurman, R.G., 2000. Estrogen is involved in early alcohol-induced liver injury
in a rat enteral feeding model. Hepatology 31, 117123.