Journal of Hepatology 1997;21: 363-310
Printed in Denmark AN rights reserved
MunksgnardCopenhagen
Antioxidant enzyme status in biliary obstructed rats: effects of
N-acetylcystee
Ana Pastor, Pilar S. Collado,
‘Department of Physiology,
Background/Aims: N-acetylcysteine (NAC) is a
modulator of thiol levels that protects against hepato-
toxic agents. The aim of this study was to investigate
whether NAC might improve hepatic antioxidant de-
fenses in chronically biliary obstructed rats.
Methods: Secondary biliary cirrhosis was induced by
28 days of bile-duct obstruction. Groups of control
and cirrhotic animals received NAC (50 ~01
- kg-’ - d-’ i.m.) through the experimental period.
Results: Bile-duct obstruction resulted in decreased
liver glutathione concentrations. Dichlorofluorescein
(DCF) and thiobarbituric acid reactive substances
(TBARS) concentrations, measured as markers of
production of reactive oxygen species and lipid peroxi-
dation, respectively, were significantly increased.
Microsomal and mitochondrial membrane fluidity and
the activities of catalase, cytosolic and mitochondrial
superoxide dismutase (SOD), glutathione S-transfer-
B CIRRHOSIS due to common bile duct ob-
ILIARY
struction is a liver disorder characterized by im-
paired hepatic function, portal hypertension and some
additional complications that cause high perioperative
mortality (1). New therapeutic approaches should be
evaluated with the aim of improving survival and dim-
inishing liver injury.
It is known that bile-duct ligation results in a shift
in the antioxidant/prooxidant balance in favor of in-
creased free radical activity (2) and that decreased ac-
tivity of the liver mitochondrial electron transport
chain shows a correlation with lipid peroxidation (3).
The enhancement of antioxidant defenses has been
Received 15 July 1996; revised 24 February; accepted 7 March 1997
Correspondence: Javier Gonzalez-Gallego, Departamento
de Fisiologia, Farmacologia y Toxicologia, Universidad
de Leon, Campus Universitario, 24071 Lecn, Spain. Tel:
34 87 291258. Fax: 34 87 291267. e-mail dftjgg@uni-
leon.es
Copyright 0 European Associalm
for the Srudv of the Liver 1997
Journalof Hepatology
ISSN 0168-8278
M. Almar and Javier Gonzalez-Gallego
Pharmacology and Toxicology, University of Ledn, Spain
ase, and cytosolic and mitochondrial Se-dependent
and Se-independent glutathione peroxidase (GPx)
were significantly reduced. NAC corrected the reduc-
tion in glutathione concentration and partially pre-
vented the increases in DCF and TBARS concen-
trations. In addition, NAC treatment resulted in sig-
nificant preservation of membrane fluidity and of the
activities of catalase, mitochondrial SOD and the dif-
ferent forms of GPx.
Conclusions: Our data indicate that NAC maintains
antioxidant defenses in biliary obstructed rats. These
effects of NAC suggest that it may be a useful agent
to preserve liver function in patients with biliary ob-
struction.
Key words: Biliary obstruction; Catalase; Glutathi-
one; N-acetylcysteine; Peroxidase; Superoxide dismu-
tase; Transferase.
proposed to protect against some of the clinical mani-
festations of obstructive jaundice (4). In fact, it has
recently been reported that S-adenosylmethionine re-
duces lipid peroxidation and normalizes Na+/K+ ATP-
ase in bile-duct-obstructed rats (5). Our own results in-
dicate that S-adenosylmethionine administration par-
tially preserves microsomal function in secondary bili-
ary cirrhosis (6).
Modulation of cellular thiols has been used to pro-
tect the liver and hepatocytes against attack by reactive
oxygen intermediates and is currently being investi-
gated as a novel therapeutic strategy in different liver
pathologies. One of the most extensively studied agents
is N-acetylcysteine (NAC), a drug with multiple thera-
peutic applications (7,8). NAC has been shown to im-
prove tissue oxygenation in patients with fulminant
liver failure (9) to exert a protective effect against hep-
atic ischemia-reperfusion injury (10) and to improve
hepatic microcirculation after orthotopic liver trans-
plantation (11). NAC has also been reported to reduce
363
A. Pastor et al.

TABLE 1 increasing the free radical scavenging capacity of the

Effects of bile-duct obstruction (BDO) and NAC on liver and spleen liver might help to improve hepatic function in biliary
weight, mortal txessure, and serum AST activity
obstruction.
Control NAC BDO BDO+NAC Mammalian cells possess different enzymatic anti-
Liver weight (g) 14.OkO.5 12.920.5 20.8~1.3 21.1k11.6’ oxidant defenses to cope with oxygen free radicals, in-
Spleen weight (g) 0.86CO.05 0.75+0.07 2.28t0.18’ 1.75f0.212,3
cluding superoxide dismutase (SOD), catalase, gluta-
Portal pressure 6.9kO.3 6.6-cO.2 11.3%0.5’ 9.8k0.32,3
(mmHg)
thione peroxidase (GPx) and glutathione S-transferase
AST (U/I) 107z13 9627 35O-t60’ 228k432,3 (15). Systematic investigations assessing antioxidant
Rats were duct ligated or sham operated as described in Materials enzyme status or its protection by thiol modulators in
and Methods. Results are the mean%SEM for 6 to 12 rats. ‘pt0.05 bile-duct obstruction have not been carried out until
significantly different from control. *p<O.O5 significantly different
from NAC. 3 (0.05 significantly different from BDO.
now. The purpose of this study was to investigate the
AST=aspartate aminotransferase. effects of biliary obstruction on the activity of antioxi-
dant enzymes in the liver and to ascertain whether
NAC treatment exerts any beneficial effect on antioxi-
dant defenses in chronically obstructed rats.
liver damage resulting from paracetamol overdosing in
humans (12) and to diminish liver injury and prevent
liver and plasma glutathione depletion in mice (13). Materials and Methods
NAC is a sulfhydryl group donor serving as a precur- Materials
sor of glutathione synthesis (14) and inhibits the for- N-acetylcysteine, 1-chloro-2,4_dinitrobenzene, cumene
mation of extracellular reactive oxygen intermediates hydroperoxide, epinephrine, 1,6-diphenyl- 1,3&hexatri-
(10). In the light of these findings it is reasonable to ene, glutathione reductase, nicotinamide adenine dinu-
expect that NAC administration, by safeguarding or cleotide phosphate, 0-phthalaldehyde and reduced glu-

1
qControl
QNAC
qBDO
qBDO+NAC

Fig. I. Effect of bile-duct obstruction (BDO) and N-acetylcysteine (NAC) on glutathione concentration in liver homogenates
and on liver dichlorofluorescein (DCF) and thiobarbituric acid reactive substances (TBARS) concentrations. Values are
means?s.e.mean (SEM) for 6 to 12 rats. *p<O.O5 signz&antly different from control. Jp<O.OS significantly different from
NAC. #p<O.O5 sign$cantly different from BDO.

364
 Antioxidant enzyme status and NAC in biliary obstruction

tathione were purchased from Sigma Chemical Co. (St qControl

Louis, MO, USA). 2’-7’-dichlorofluorescein diacetate
was obtained from Molecular Probes (Eugene, OR,
USA). All other reagents were of the highest quality QBDO+NAC
commercially available.
0.24 1

Animals and experimental procedures

Male Wistar rats were obtained from Charles River
(Barcelona, Spain). They were kept on standard rat
chow (Purina Chow A03; Panlab Ltd, Barcelona,
Spain) with free access to tap water, in temperature-
and humidity-controlled animal quarters with a 12-h
light-dark cycle. Secondary biliary cirrhosis was in-
duced in animals weighing around 270 g by double lig-
ation and division of the common bile duct (16,17).
The animals were sacrificed after 28 days of obstruc-
tion, a period at which there is a complete development
0.24,
of both cholestasis and cirrhosis, and liver biochemical
changes are clearly established (6,16). Control animals
were sham operated.
Groups of control and cirrhotic animals received
daily i.m. injections of NAC (50 pmollkg in 0.5 ml of
0.154 M NaCl) through the experimental period.
Other groups received daily equivalent volumes of the
solvent. The dose of NAC was equimolecular with that
of S-adenosylmethionine previously reported to reduce
liver injury in biliary-obstructed rats (5).

Tissue preparation Fig. 2. Effect of bile-duct obstruction (BDO) and NAC on

To eliminate diurnal effects, all rats were killed at the
same time of the day. After the animals were decapitated
and exsanguinated, the livers and spleens were immedi-
ately removed. A lobe of the liver was frozen in liquid
nitrogen. Another lobe was homogenized in an ice-cold
medium containing 250 mM mannitol, 70 mM sucrose,
and 2 mM EDTA (pH 7.4) for preparing mitochondria
and cytosol. The homogenate was centrifuged at 4°C
and 750 g for 10 min to pellet the nucleus, red cells and
cell debris fractions. Mitochondria were isolated by cen-
trifugation at 12000 g for 10 min of the supernatants.
The precipitate was resuspended and again centrifuged.
The mitochondrial pellet was diluted to contain ap-
proximately 10 mg mitochondrial protein per ml. The
supernatant fraction of the 12000 g centrifugation of
the liver homogenate was centrifuged again at 105 000 g
for 60 min, and the supernatant was retained as cytosol.
All prepared subcellular fractions were frozen at - 80°C
until assays were performed.
Analytical methods
All mitochondrial enzymes were assayed after frozen
and thawed three times. SOD (EC 1.15.1 .l) was as-
sayed according to Misra & Fridovich (18) at 30°C.
fluorescence polarization, an expression of membrane ju-
idity, in microsomal and mitochondrial membranes. Values
are means?SEM for 6 to 12 rats. *p<O.O5 significantly
different from control. 3 p<O.OS significantly different from
NAC. #p<O.O5 signljicantly different from BDO. Abbrevi-
ations as in Fig. 1.
The amount of enzyme that causes 50% inhibition of
epinephrine autooxidation is defined as 1 unit. Cata-
lase (EC 1.11.1.6) activity was measured by the
method of Chance & Machley (19). The assay of GPx
(EC 1.11 .1.19) was carried out according to Flohe &
Gunsler (20). H202 and cumene hydroperoxide were
used as substrates to measure the Se-dependent and
total GPx activities, respectively. The difference be-
tween the two was designated as the Se-independent
GPx activity. Glutathione S-transferase (EC 2.5.1.18)
was determined in cytosol at 25°C according to
Habig et al. (21) using 1-chloro-2,4_dinitrobenzene as
substrate. Glutathione analysis was performed fluor-
imetrically by the method of Hissin & Hilf (22). Flu-
idity of microsomal and mitochondrial membranes
was assayed by measurement of steady-state fluor-

365
A. Pastor et al.

escence polarization of 1,&diphenyl- 1,3,5_hexatriene
incorporated into the hydrophobic core of the mem-
brane layer using a SLM-Aminco spectrofluorimeter
(model LH-700; SLM Instruments, Urbana, IL,
USA) equipped with polarizers in both excitation and
emission beams. The fluorescence intensity was meas-
ured perpendicular (IJ and parallel (Ii,) to the polar-
ization phase of the exciting light. The steady-state
fluorescence polarization (P) was calculated according
to the ratio p=(&,-It)/(Ip+It) (ref. 23). Thiobarbituric
acid reactive substances (TBARS) were determined in
liver homogenates according to Ohkawa et al. (24).
Production of reactive oxygen species (ROS) was
monitored with a fluorescence probe, 2’-7’-dichloro-
fluorescein (DCF). Fluorescence was determined at
550 nm for emission and 470 nm for excitation, re-
spectively, according to the spectral characteristics of
DCF (25). Protein concentration was measured ac-
cording to Lowry et al. (26), using serum bovine
albumin as standard. The serum activity of aspartate
aminotransferase (AST) was estimated using a com-
mercially available kit (Boehringer Mannheim, Ger-
many). Portal pressure was measured after cannulat-
ing the portal vein with a 21-gauge needle connected
to a manometer filled with saline. The height of the
right atrium was taken as the zero reference level (27).
Statistical analysis
Means and SEMs were calculated for all data. Signifi-
cant differences between means were evaluated by
analyses of variance. Post hoc comparisons between
the different groups were done by the Newman-Keuls
test. A difference was considered significant when p
was less than 0.05.
Results
Characterization of’ the rats
The general characteristics of the rats used in the cur-
rent study are shown in Table 1. Bile-duct obstruction
resulted in significant increases in liver weight, portal
pressure and the serum activity of AST. Spleen weight,
as an expression of portal hypertension, was also sig-

2.5
1

Fig. 3. Effect of bile-duct obstruction (BDO) and NAC on liver catalase, glutathione S-transferase and cytosolic and mito-
chondrial SOD activities. Values are means?SEM for 6 to 12 rats. *p<O.O5 significantly dijjferent from control. jp<O.O5
significantly different from NAC. #p<O.O5 sign@antly dljjferent from BDO. Abbreviations as in Fig. 1.

366
 Antioxidant enzyme status and NAC in biliary obstruction

qControl
NNAC
BDO
•JBDO+N.~~

Fig. 4. Effect of bile duct obstruction (BDO) and NAC on liver cytosolic and mitochondrial Se-dependent GPx or Se-
independent glutathione peroxidase (GPx) activities. Values are means?SEM for 6 to 12 rats. *p<O.O5 significantly dtffer-
ent from control. jp<O.O5 significantly dtff erent from NAC. # p<O.OS significantly dtff erent from BDO. Abbreviations as in
Fig. 1.

nificantly elevated. NAC treatment partially prevented
the increase in spleen weight and the change in portal
pressure was partially corrected. The elevation of AST
activity was significantly attenuated by NAC adminis-
tration (Table 1).
Effects of NAC on liver glutathione, production of
ROS and lipid peroxidation
The glutathione concentration was significantly re-
duced in liver homogenates after 28 days of biliary ob-
struction. NAC treatment normalized glutathione con-
centration to levels comparable to those in control ani-
mals (Fig. 1). DCF and TBARS concentrations were
determined in liver homogenates as markers of ROS
production and lipid peroxidation, respectively. As
shown in Fig. 1, bile-duct ligation resulted in an in-
crease in DCF and TBARS concentrations. These ef-
fects were attenuated by NAC treatment, although
values were still significantly higher than those found
in the controls (Fig. 1).
Effects of NAC on thejuidity of membranes
As shown in Fig. 2, steady-state fluorescence polariza-
tion anisotropy, a marker of membrane fluidity, was
significantly increased, both in microsomal and mito-
chondrial membranes, indicating a more rigid mem-
brane structure in animals with biliary cirrhosis. The
decrease in microsomal and mitochondrial membrane
fluidity was prevented by NAC treatment.
Effects of NAC on antioxidant enzyme status
Biliary obstruction caused a marked reduction in the
activities of catalase, cytosolic and mitochondrial
SOD, glutathione S-transferase, cytosolic and mito-
chondrial Se-dependent GPx and cytosolic and mito-

367
A. Pastor et al.
chondrial Se-independent GPx (Fig. 3 and 4). Admin-
istration of NAC to bile-duct-obstructed rats increased
glutathione S-transferase activity to values similar to
controls (Fig. 3). NAC partially prevented the decrease
in the activities of catalase, mitochondrial SOD, and
cytosolic and mitochondrial Se-dependent and Se-in-
dependent GPx (Fig. 3 and 4). Cytosolic SOD activity
was unaffected by NAC treatment (Fig. 3).
Discussion
Previous studies have shown increased concentrations
of TBARS and a reduction in some antioxidant de-
fenses in rats with bile-duct ligation, suggesting the
presence of oxidative stress and a shift in the prooxi-
dant/antioxidant balance in favor of lipid peroxidation
(2). More recently it has been found that the concen-
trations of glutathione and ubiquinone are decreased
in liver mitochondria from bile-duct-ligated rats, in as-
sociation with increased concentration of TBARS (3).
Increased oxygen radical production in obstructed rats
could be explained by the hepatic accumulation of
lipophilic bile acids (28) and endotoxinemia (29), fac-
tors that would impair the activity of the electron
transport chain in liver mitochondria and thus, in turn,
further increase the production of reactive oxygen in-
termediates (3,30).
Our results confirm that secondary biliary cirrhosis
develops with increased generation of reactive oxygen
species, as reflected in DCF, and production of lipid
peroxidation as TBARS. Although radicals may inter-
act with virtually any cell structure, polyunsaturated
fatty acids are specially susceptible. As lipid peroxi-
dation occurs, oxidative damage of membrane lipids
(31) may alter the functional integrity of membranes.
This is supported by the finding of increased fluor-
escence polarization in both microsomal and mito-
chondrial membranes of obstructed rats. It has been
shown previously that biliary cirrhosis in rats de-
velops with marked alterations in the composition of
microsomal membranes (32), a fact further confirmed
by spin label methods (33). In addition to membrane
lipid deterioration, free radical production causes
changes in membrane protein structure (34), and both
factors may lead to alterations in membrane-
bound enzyme activities such as Na+/K+ ATPases
(5) cytochrome P450-dependent monooxygenases (6)
and complexes of the electron transport chain (3).
Different mechanisms may contribute to a reduced
activity of antioxidant enzymes in biliary-obstructed
rats. These protective enzyme systems are known to
be inhibited by radical reaction products. For instance,
SOD is inhibited by hydrogen peroxide (35), and both
catalase and GPx are inhibited by superoxide (36). Ad-
368
ditionally, rats with biliary obstruction lack vitamin E
and selenium (2). Selenium deficiency is known to
cause depletion of tissue GPx activity, making tissues
more vulnerable to oxidative damage (37). Inhibition
by bilirubin accumulated in the liver might contribute
to the impairment in the activity of glutathione S-
transferase in bile-duct-obstructed rats (38). Whatever
the mechanism responsible, inadequate activity of free
radical scavenging enzymes could initiate a vicious
cycle by increasing free radical production, thereby ex-
ceeding the antioxidant liver capacity and resulting in
further oxidative damage.
NAC treatment prevented the decrease in liver gluta-
thione concentration. Although glutathione depletion
is not necessarily sufficient to cause lipid peroxidation,
it is agreed that it may favor the peroxidation produced
by other factors (39). It has been found that glutathi-
one is depleted in carbon tetrachloride-cirrhotic rats,
leading to SAM synthetase inactivation (40). Adminis-
tration of glutathione precursors, such as S-adenosyl-
methionine, might break this vicious circle by produc-
ing increased glutathione levels that could help to pro-
tect the cells against the effects of free radicals (39,40).
The increase in TBARS levels induced by biliary ob-
struction in our experiments was partially prevented by
NAC treatment, while liver glutathione concentration
normalized. Since NAC metabolism is related to re-
duced glutathione, at least part of the beneficial effects
of NAC may be linked to the inhibition of lipoperoxi-
dative processes.
Restoration of membrane properties and mitochon-
drial function would further contribute to the protec-
tive effect of NAC. It has been shown that both S-
adenosylmethionine and NAC prevent the inhibition
by ethanol of rat liver plasma membrane Na+/K+
ATPase (41), a fact that has been related to the main-
tenance of the glutathione pool. NAC has been re-
ported to partially normalize cytosolic and mitochon-
drial glutathione levels in rats given phorone, restoring
important mitochondrial functions (42). Exposure of
isolated mouse hepatocytes to toxic concentrations of
paracetamol is associated with decreases in ATP levels,
ATP/ADP ratio and lactate/piruvate ratio. NAC pre-
vents these effects by reducing the extent of plasma
membrane damage and protecting against the impair-
ment of cellular respiration (43).
Another possible mechanism of action of NAC
may also improve liver function. NAC effects on pa-
tients with fulminant liver failure have led to the
hypothesis that the replenishment of liver sulfhydryl
groups could improve the ability of liver cells to ex-
tract and use oxygen (9). However, this effect is ap-
parently absent in ischemia-reperfusion injury (10)

and, although administration of NAC results in in-
creased oxygen delivery, it does not confer hemody-
namic benefits on patients with cirrhosis (44). Other
authors have proposed that NAC protects against is-
chemia-reperfusion injury by inhibiting reactive oxy-
gen intermediates production by Kupffer cells (10).
The possibility of Kupffer cell activation in bilary ob-
struction requires to be explored.
In summary, secondary biliary cirrhosis in the rat
develops with glutathione depletion, lipid peroxidation
and impaired antioxidant enzyme status. Our data
show that NAC reduces lipid peroxidation and main-
tains protective levels of antioxidant enzymes in bili-
ary-obstructed rats. These effects of NAC suggest that
it may be useful in preserving liver function in patients
with biliary obstruction.
Acknowledgements
Ana Pastor was partially supported by a grant from
Europharma (Boehringer Ingelheim, Spain).
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