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Role of nitric oxide in hepatic microvascular injury
elicited by acetaminophen in mice
Yoshiya Ito, Edward R. Abril, Nancy W. Bethea, and Robert S. McCuskey
Department of Cell Biology and Anatomy, College of Medicine, University of Arizona, Tucson, Arizona 85724-5044
Submitted 12 May 2003; accepted in final form 9 September 2003
Ito, Yoshiya, Edward R. Abril, Nancy W. Bethea, and Robert S.
McCuskey. Role of nitric oxide in hepatic microvascular injury
elicited by acetaminophen in mice. Am J Physiol Gastrointest Liver
Physiol 286: G60–G67, 2004. First published September 11, 2003;
10.1152/ajpgi.00217.2003.—Nitric oxide (NO) is suggested to play a
role in liver injury elicited by acetaminophen (APAP). Hepatic mi-
crocirculatory dysfunction also is reported to contribute to the devel-
opment of the injury. As a result, the role of NO in hepatic microcir-
culatory alterations in response to APAP was examined in mice by in
vivo microscopy. A selective inducible NO synthase (iNOS) inhibitor,
L-N
6
-(1-iminoethyl)-lysine (L-NIL), or a nonselective NOS inhibitor,
N
G
-nitro-L-arginine methyl ester (L-NAME), was intraperitoneally
administered to animals 10 min before APAP gavage.
L-NIL sup-
pressed raised alanine aminotransferase (ALT) values 6 h after APAP,
whereas
L-NAME increased those 1.7-fold. Increased ALT levels
were associated with hepatic expression of iNOS.
L-NIL, but not
L-NAME, reduced the expression. APAP caused a reduction (20%) in
the numbers of perfused sinusoids.
L-NIL restored the sinusoidal
perfusion, but
L-NAME was ineffective. APAP increased the area
occupied by infiltrated erythrocytes into the extrasinusoidal space.
L-NIL tended to minimize this infiltration, whereas L-NAME further
enhanced it. APAP caused an increase (1.5-fold) in Kupffer cell
phagocytic activity. This activity in response to APAP was blunted by
L-NIL, whereas L-NAME further elevated it. L-NIL suppressed
APAP-induced decreases in hepatic glutathione levels. These results
suggest that NO derived from iNOS contributes to APAP-induced
parenchymal cell injury and hepatic microcirculatory disturbances.
L-NIL exerts preventive effects on the liver injury partly by inhibiting
APAP bioactivation. In contrast, NO derived from constitutive iso-
forms of NOS exerts a protective role in liver microcirculation against
APAP intoxication and thereby minimizes liver injury.
sinusoids; endothelial cell; Kupffer cell; hemorrhage
ACETAMINOPHEN, ALSO KNOWN AS paracetamol (N-acetyl-para-
aminophenol; APAP), is a commonly used over-the-counter
analgesic and antipyretic with few side effects when taken at
therapeutic doses. However, APAP intoxication from overdos-
ing can result in severe hepatic damage, which is characterized
by hemorrhagic centrilobular (CL) necrosis and by towering
the levels of transaminase in both humans and animals (34).
Recently, it has been shown that nitric oxide (NO) is involved
in the progression of APAP-induced liver injury. APAP hep-
atotoxicity is associated with increased expression of inducible
NO synthase (iNOS) (7, 33) and the formation of nitrotyrosine-
protein adducts (12, 19). Inhibition of iNOS with aminogua-
nidine is reported to minimize liver injury elicited by APAP (7,
8). However, others (11) have reported that NOS inhibitors
aggravated the injury.
The APAP-induced hepatic necrosis is preceded by CL
microvascular congestion due to collapse of the sinusoidal wall
and the infiltration of blood elements into the space of Disse
(25, 36). That APAP injures sinusoidal endothelial cells
(SECs) is reflected in the appearance of large gaps in their
cytoplasm (25, 36). These findings suggest that, in addition to
direct hepatocellular damage through metabolic activation of
APAP, hepatic microcirculatory disturbance participates in
liver injury elicited by APAP overdose. We have recently
shown (15) that hepatic microvascular derangement contrib-
utes to the development of APAP hepatotoxicity by using in
vivo microscopy. Initial events occurring in the hepatic micro-
vasculature following APAP included SEC injury exhibited by
the penetration of erythrocytes into the extrasinusoidal space.
NO plays a critical role in maintaining ample blood supply
into the hepatic microvasculature by affecting the expression of
adhesion molecules on leukocytes, platelets, and endothelial
cells (20). The NOS inhibitors exacerbated the hepatic micro-
vascular inflammatory responses including leukocyte-endothe-
lial interaction to endotoxin (30) and ischemia-reperfusion
(13), suggesting that NO plays a protective role in stabilizing
the hepatic microcirculation. However, little is known about
the involvement of NO in hepatic microvascular injury elicited
by APAP. As a result, the present study was conducted to
examine the effects of NOS inhibition on hepatic microvascu-
lar injury after APAP administration by using in vivo micro-
scopic methods (24).
MATERIALS AND METHODS
Experimental animals. Male C57BL/6 mice (7–8 wk of age),
weighing 23–25 g, were purchased from Charles River Laboratories
(Charles River, Windham, ME) and were used for these experiments.
All animals were allowed free access to food and water and were
fasted 24 h before the experiments. The present study was performed
in adherence to the National Institutes of Health guidelines for the use
of experimental animals and followed a protocol approved by the
Animal Care and Use Committee of the University of Arizona. APAP
(300 mg/kg liquid Tylenol; McNeil-PPC, Ft. Washington, PA) was
given to mice by oral gavage. N
G
-nitro-L-arginine methyl ester (L-
NAME; Sigma, St. Louis, MO; 10 mg/kg body wt in 0.1 ml PBS),
which has ⬎30-fold selectivity for constitutive NO synthase (cNOS)
compared with that for iNOS (10), and
L-N
6
-(1-iminoethyl)-lysine
(L-NIL; Cayman Chemical, Ann Arbor, MI; 1, 3, and 10 mg/kg body
wt in 0.1 ml PBS), an inhibitor with 30- to 100-fold selectivity toward
iNOS compared with cNOS (29), were intraperitoneally administered
to animals 10 min before APAP gavage. Animals receiving the same
amount of vehicle (PBS) served as controls. Some of the animals were
treated with
L-NIL (10 mg/kg) 10 min before or simultaneously with
an intraperitoneal injection of APAP (300 mg/kg; Sigma). In some
Address for reprint requests and other correspondence: R. S. McCuskey,
Dept. of Cell Biology and Anatomy, College of Medicine, P.O. Box
245044, Univ. of Arizona, Tucson, AZ 85724-5044 (E-mail:
mccuskey@email.arizona.edu).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Gastrointest Liver Physiol 286: G60–G67, 2004.
First published September 11, 2003; 10.1152/ajpgi.00217.2003.
0193-1857/04 $5.00 Copyright
©
2004 the American Physiological Society http://www.ajpgi.orgG60
experiments, the NO donor S-nitro-N-acetylpenicillamine (SNAP;
Cayman) (6) was administered (20 g/kg ip) to mice 20 min before
APAP gavage.
In vivo microscopy. Animals were anesthetized with an intraperi-
toneal injection of 50 mg/kg pentobarbital sodium (Nembutal; Abbott
Laboratories, Abbott Park, IL). The hepatic microvascular responses
were examined by using established high-resolution in vivo micro-
scopic methods (24). Briefly, a compound binocular microscope
(Leitz, Wetzlar, Germany) adapted for in vivo microscopy was
equipped to provide either transillumination or epi-illumination as
well as video microscopy by using a charge-coupled device camera
(MTI, Michigan City, IN). The liver was exteriorized through a left
subcostal incision and positioned over a window of optical-grade mica
in a specially designed tray mounted on a microscopic stage. The tray
provided for the drainage of irrigating fluids, and the window overlaid
a long-working-distance condenser. The liver was covered by a piece
of Saran Wrap (Dow Chemical, Midland, MI), which held it in
position and limited movement. Homeostasis was ensured by a con-
stant suffusion of the organ with Ringer solution maintained at body
temperature. With the ⫻80–1.0 numerical aperture water immersion
objective (Leitz) employed for these studies, the methods permitted
visualization of the cells comprising the sinusoidal lining, the formed
elements of the blood, the nuclei, nucleoli, mitochondria and fat
droplets in hepatic cells, and bile canaliculi. Under optimal conditions,
the resolution was 0.3–0.5 m. Microvascular events were observed
and recorded for at least 30 s for subsequent offline analysis by using
a Sony Betacam video tape recorder (Sony Medical Electronics, Park
Ridge, NJ).
Kupffer cell (KC) function was assessed by measuring the phago-
cytosis of fluorescent 1.0-m latex particles (Fluoresbrite-fluorescent
monodispersed polystyrene microspheres; Polysciences, Warrington,
PA) by individual cells. The latex was diluted 1:10 with sterile saline
and injected in 0.1-ml volume through a mesenteric vein by using a
30-gauge lymphangiography needle (Becton Dickinson, Franklin
Lakes, NJ). The distribution and relative number of phagocytic KCs
was measured by counting the number of cells that phagocytosed latex
particles in a standardized microscopic field 15 min after each mouse
had received the injection of latex. To assess regional distribution, the
number of phagocytic KCs per microscopic field was counted in 10
periportal (PP) and 10 CL regions in each animal. The relative
adequacy of blood perfusion through the sinusoids was evaluated by
counting the number of sinusoids containing blood flow (SCF) in the
same 10 microscopic fields in which the numbers of phagocytic KCs
were determined. Because reduced perfusion of individual sinusoids
can limit the delivery of the latex particles to KCs in these vessels, the
ratio of KCs that phagocytosed latex particles to SCF was used as an
overall index of KC phagocytic activity.
To examine the interaction of leukocytes with the sinusoidal wall,
quantification of leukocytes adhering to the endothelial lining of
sinusoids was calculated by counting the number of leukocytes per
unit length of sinusoid (adherent leukocytes/100 m) in the same
microscopic fields. A leukocyte was defined as adhering to the
sinusoidal wall if it remained stationary for at least 30 s. Endothelial
swelling, which is thought to be an indication of activation and/or
injury, was assessed by counting the numbers of swollen cells whose
nuclear regions protruded across one-third or more of the lumen in the
same microscopic fields. The results were averaged, and the data were
represented as the average number in each animal.
To quantify the extent of hemorrhage elicited by APAP gavage, the
area occupied by extrasinusoidal erythrocytes was measured in the
same microscopic fields by using a computer-assisted digital imaging
processor (Scion Image; Scion, Frederick, MD). The results were
expressed as extrasinusoidal area occupied by erythrocytes (m
2
/10
CL regions). In addition, the diameters of SCF were measured in the
same microscopic fields from video recordings.
Sampling and assays. In a separate set of experimental animals,
blood was collected from the inferior vena cava and the samples were
separated by centrifugation at 13,000 g for 5 min at 4°C. The samples
were stored at ⫺70°C until assays were performed. The serum
activities of alanine aminotransferase (ALT) were measured by enzy-
matic procedures by using a diagnostic kit (Sigma). In addition, the
serum concentrations of nitrite/nitrate were determined with a kit
(Cayman) by means of Greiss reaction.
Histology and immunohistochemical studies. The hepatic tissues
were immediately fixed with 4% paraformaldehyde in 0.1 M phos-
phate buffer solution (pH 7.4). After fixation, the tissues were dehy-
drated with a graded series of ethanol solutions and embedded in
paraffin. The sections (5 m) from the paraffin-embedded tissues
were stained with hematoxylin and eosin. For immunostaining, the
sections were incubated first with normal goat serum and then with
rabbit anti-murine iNOS antibody (1:100 dilution; Santa Cruz Bio-
technology, Santa Cruz, CA) or with anti-nitrotyrosine antibody
(1:100 dilution; Molecular Probes, Eugene, OR) for 1 h. After being
washed, the sections were incubated with biotinylated anti-rabbit IgG
followed by incubation with avidin-biotin-peroxidase complex (Dako
kit; DakoCytomation, Carpinteria, CA). Reaction products were vi-
sualized by treating sections with 0.02% 3,3⬘-diaminobenzidine and
0.3% nickel ammonium sulfate in 50 mM Tris䡠 HCl buffer (pH 7.4).
The sensitivity of the reaction was verified by using isotype-matched
immunoglobulin (rabbit immunoglobulin; Sigma) in the same man-
ner. All steps were performed at room temperature.
RT-PCR analysis. Two and six hours after APAP gavage, ⬃100 mg
of the liver tissue were excised and stored at ⫺20°C until being used.
Total cellular RNA was extracted from the tissue by using a commer-
cially available kit (SV total RNA isolation system; Promega, Mad-
ison, WI) in accordance with the manufacturer’s instructions. Hepatic
iNOS mRNA was determined by means of semiquantitative RT-PCR
(PCR ELISA kit; Roche Diagnostics, Basel, Switzerland). Amplica-
tion was performed as follows: 94°C, 45 s; 54°C, 45 s; and 72°C, 45 s
for iNOS and -actin, followed by a final extension for 10 min at 72°C
and by soak at 4°C with the addition of 62.5 M digoxigenin
(DIG)-dUTP for PCR labeling. To avoid tube-to-tube variation, the
primers for -actin and iNOS were added together to each reaction.
The two cDNA were coamplified. One microliter of the PCR products
was denatured and incubated with commercially biotinylated probes
(murine -actin: 5⬘-GGGTGTTGAAGGTCTCAAACATGATCT-
GGG-3⬘; murine iNOS: 5⬘-TCTTGGGTCTCCGCTTCTCGTC-3⬘)
for4hat37°C on a shaker in a streptavidin-coated 96-well plate.
Peroxidase-conjugated anti-DIG antibody was added and was mea-
sured by an ELISA reader. Quantitative determination of mRNA was
calculated based on the slope of the standard curve and was expressed
in attomoles per milliliter.
Measurement of hepatic total glutathione. Hepatic total glutathione
(GSH) was determined colorimetrically by using a commercially
available kit (Oxford Biomedical Research, Oxford, MI). Briefly, a
portion of liver tissue was homogenized in 10 ml of cold 5%
metaphosphoric acid and was centrifuged at 3,000 g for 10 min. The
upper aqueous layer was collected and assayed according to the
manufacturer’s protocol.
Statistical analysis. All data were expressed as means ⫾ SE.
Multiple comparisons were performed by using one-way ANOVA
with a post hoc Fisher’s test. Differences were considered to be
significant at P ⬍ 0.05.
RESULTS
Effect of NOS inhibitors on the levels of ALT after APAP
gavage. Figure 1 shows the levels of ALT activities after oral
gavage with APAP. The levels of ALT 2 h after APAP gavage
did not change significantly. Neither L-NIL nor L-NAME
significantly changed ALT levels in mice treated with APAP.
At 6 h after APAP, the levels of ALT were markedly increased.
L-NIL decreased the ALT levels by 90% in a dose-dependent
manner, whereas L-NAME further elevated them. Both L-NIL
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and L-NAME by themselves resulted in no significant changes
in ALT levels (data not shown). On the basis of these results,
we selected a dose of 10 mg/kg L-NIL for the remaining
studies. In addition, the intraperitoneal administration of APAP
caused a significant increase in ALT levels 6 h after APAP
(1,480 ⫾ 193 IU/l; n ⫽ 5). Pretreatment with L-NIL (10 mg/kg)
significantly reduced those (824 ⫾ 107 IU/l; n ⫽ 5), whereas
cotreatment with L-NIL and APAP did not significantly change
them (977 ⫾ 109 IU/l; n ⫽ 5).
Effects of NOS inhibitors on liver microcirculation in re-
sponse to APAP. Figure 2 illustrates the effects of L-NIL and
L-NAME on hepatic microvascular responses to APAP. The
numbers of SCF in CL regions were significantly decreased 2 h
after APAP when compared with untreated controls. L-NIL, but
not L-NAME, restored sinusoidal perfusion (Fig. 2A). At 6 h
after, the numbers of SCF in PP and CL regions were reduced
by 9 and 21%, respectively. Also, L-NIL, but not L-NAME,
improved the SCF. The oral gavage with APAP caused a
significant increase in KC phagocytic activity in both PP and
CL regions 2 and 6 h after APAP (Fig. 2B). L-NAME further
raised KC phagocytic activity after APAP, whereas L-NIL
inhibited this activity. APAP failed to induce any changes in
the numbers of leukocytes adhering to the sinusoids in both PP
and CL regions as well as to the central venules 2 and 6 h after
APAP (not shown). The numbers of swollen SECs in PP
regions were significantly increased (2.8-fold) 2 and 6 h after
APAP. Neither L-NIL nor L-NAME changed the numbers of
SECs in APAP-treated mice. Although there was no evidence
of infiltrated erythrocytes into the space of Disse (namely,
hemorrhage) 2 h after APAP gavage, infiltration was observed
6 h after APAP (612 ⫾ 362 m
2
/10 CL regions). L-NAME
elicited hemorrhage as early as 2 h after APAP (108 ⫾ 89
m
2
/10 CL regions), although the hemorrhagic area was small.
At 6 h after APAP, the hemorrhagic regions in
L-NAME-
treated mice (2,396 ⫾ 1,173 m
2
/10 CL regions) exceeded
those in vehicle-treated mice. The diameters of PP sinusoids
did not change significantly in each experimental group (not
shown). In contrast, those of CL sinusoids were significantly
decreased 2 and 6 h after APAP (6.7 and 12.4%, respectively).
L-NAME did not change the diameters in APAP-treated mice,
whereas L-NIL inhibited the reduced diameters.
Effects of NOS inhibitors on iNOS and nitrotyrosine expres-
sion in the liver treated with APAP. To elucidate the role of NO
in APAP liver injury, we determined the hepatic expression of
iNOS and nitrotyrosine. The expression of hepatic iNOS
mRNA was upregulated 2 h (5.6-fold) and 6 h (9.3-fold) after
APAP, respectively, when compared with controls (Fig. 3).
L-NIL suppressed the increased expression of iNOS mRNA 6 h
after APAP by 56.2%, whereas L-NAME did not change
significantly.
Fig. 1. Effect of L-N
6
-(1-iminoethyl)-lysine (L-NIL) or N
G
-
nitro-
L-arginine methyl ester (L-NAME) on serum activities of
alanine aminotransferase (ALT) in mice treated with acetamin-
ophen (APAP). The levels of ALT were determined in un-
treated controls and 2 and 6 h after oral gavage with 300 mg/kg
APAP. Mice were pretreated with L-NIL (1, 3, and 10 mg/kg
ip) or L-NAME (10 mg/kg ip) 10 min before APAP. Veh,
vehicle. Data are means ⫾ SE. *P ⬍ 0.05 vs. untreated controls
(time 0).
Fig. 2. Effects of L-NIL or L-NAME on the
numbers of sinusoids containing blood flow
(SCF; A) and on Kupffer cell (KC) phago-
cytic activity (B) in mice treated with APAP.
The numbers of SCF and phagocytic KCs
were measured in 10 periportal (closed bars)
and 10 centrilobular (open bars) regions in
each animal. Mice were pretreated with L-
NIL (10 mg/kg ip) or L-NAME (10 mg/kg
ip) 10 min before APAP. Cont, control. Data
are means ⫾ SE from 6 mice per group.
*P ⬍ 0.05 vs. untreated controls (time 0).
†P ⬍ 0.05 vs. vehicle-treated mice.
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The immunoreactivity with iNOS in the liver was negative
2 h after APAP (data not shown). At 6 h after APAP, the
staining for iNOS was expressed in CL hepatocytes in the same
area where necrosis was exhibited (Fig. 4A). L-NIL and L-
NAME inhibited the expression of iNOS in CL regions (Fig. 4,
B and C). Liver sections stained with hematoxylin and eosin
from mice pretreated with L-NIL showed reduction in hepatic
necrosis after APAP gavage (Fig. 4D).
The staining for nitrotyrosine, a convenient marker of per-
oxynitrite (2), in the liver from controls was negative (Fig. 5A).
Two hours after APAP, the immunoreactivity with nitroty-
rosine was intensely expressed in the CL sinusoids and was
also shown in some individual hepatocytes in midlobular to CL
regions (Fig. 5B). Six hours after APAP, a confluent staining of
PP hepatocytes was seen, although there was no staining of the
sinusoids or hepatocytes in the CL regions (Fig. 5C). Pretreat-
ment with
L-NIL exhibited the positive staining for nitroty-
rosine in individual hepatocytes in PP regions 2 and 6 h after
APAP (Fig. 5D). L-NAME induced strong positive staining for
nitrotyrosine in the CL sinusoids 2 and 6 h after APAP (Fig. 5,
E and F).
Table 1 summarizes the serum concentrations of nitrite/
nitrate at 2 and 6 h after APAP gavage. There were no
significant differences in the levels of nitrite/nitrate among the
experimental groups.
Effect of L-NIL on total GSH levels in the liver treated with
APAP gavage. The bioactivation of APAP during the initiation
phase of the liver injury causes a significant decrease in hepatic
GSH levels, which correlate with APAP hepatotoxicity (28).
To address the question of whether L-NIL may affect APAP
bioactivation, we measured hepatic GSH levels. As shown in
Fig. 6, the total GSH levels 1 and 2 h after APAP were
decreased by 59.3 and 70.3%, respectively. L-NIL restored
GSH levels to 75% of control levels at both time points after
APAP.
Effect of NO donors on liver microcirculation after APAP
gavage. To further investigate the role of NO in liver injury in
response to APAP, the NO donor SNAP was administered to
mice treated with APAP. Table 2 summarizes the effects of
pretreatment with SNAP on ALT activity and liver microcir-
culation 6 h after APAP administration. SNAP further in-
creased ALT levels when compared with vehicle-treated mice;
however, no significant changes in liver microcirculation were
shown between SNAP-treated and vehicle-treated mice.
DISCUSSION
The results of the present study demonstrated that oral
gavage with APAP resulted in liver microcirculatory dysfunc-
tion including impaired sinusoidal perfusion, swelling of SECs,
infiltrated erythrocytes in the extrasinusoidal space, and acti-
vated KC phagocytic function (15). The liver microcirculatory
dysfunction elicited by APAP preceded parenchymal cell in-
Fig. 3. Effect of L-NIL or L-NAME on hepatic inducible nitric oxide (NO)
synthase (iNOS) mRNA expression after APAP gavage. The levels of iNOS
mRNA in the liver were determined by RT-PCR analysis. Mice were pre-
treated with L-NIL (10 mg/kg ip) or L-NAME (10 mg/kg ip) 10 min before
APAP. Data are means ⫾ SE from 3 animals per group. #P ⬍ 0.05 vs.
untreated controls (time 0). *P ⬍ 0.05 for vehicle vs. L-NIL at 6 h.
Fig. 4. Photographs of the liver sections stained with iNOS
(A, B, and C) or hematoxylin and eosin (D) 6 h after APAP
in combination with vehicle (A), L-NIL (B and D), or
L-NAME (C). A: immunoreactivity with iNOS was shown
in centrilobular hepatocytes treated with APAP. B: staining
for iNOS was negative in the centrilobular hepatocytes
pretreated with L-NIL. C: L-NAME reduced the expression
of iNOS in centrilobular hepatocytes. D: hematoxylin and
eosin-stained liver sections from mice pretreated with L-
NIL showed minimal changes. PV, portal vein; CV, central
vein. Original magnification, ⫻200.
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jury as indicated by a significant increase in ALT values and
hepatic necrosis. L-NIL, an inhibitor of iNOS, attenuated
APAP-induced hepatocellular injury as well as hepatic micro-
circulatory dysfunction. In contrast, L-NAME, a nonselective
NOS inhibitor that favors cNOS over iNOS, aggravated the
injury. In particular, L-NAME exacerbated the extrasinusoidal
area occupied by erythrocytes and KC phagocytic function in
response to APAP.
NO has been implicated in the progression of APAP hepa-
totoxicity. APAP-induced liver injury is associated with in-
creased iNOS protein expression (7, 8, 33) as well as increased
iNOS mRNA expression (14), which is consistent with our
present results. The immunohistochemical studies showed that
iNOS was expressed in the damaged hepatocytes in the CL
regions, suggesting that these hepatocytes are a source of iNOS
(7). Increased NO production from isolated hepatocytes also
has been reported (7). Furthermore, mice deficient in iNOS
moderately reduce release of hepatic enzyme after APAP
treatment (8). However, Michael et al. (26) reported that there
is no difference in the amount of hepatic necrosis between
iNOS knockout mice and wild-type mice, and Bourdi et al. (3)
reported that both iNOS knockout and wild-type mice exhib-
ited the same raised ALT levels after APAP (150 mg/kg). The
discrepancy within iNOS knockout mice still remains unclear.
Pretreatment with aminoguanidine, a selective iNOS inhibitor,
minimizes liver injury elicited by APAP (7, 8). However,
others (11) have shown that the simultaneous administration of
aminoguanidine with APAP increased the initial rate of devel-
opment of hepatotoxicity as indicated by ALT values (within
4 h after APAP), but there was no significant difference in ALT
levels 6 and 8 h after APAP. The current study also showed
that another selective iNOS inhibitor, L-NIL, attenuated APAP-
induced liver injury, which was associated with reduced ex-
pression of iNOS in the liver treated with APAP. These
findings suggest that NO derived from iNOS is involved in the
development of APAP hepatotoxicity. In contrast, Hinson et al.
(11) reported that the simultaneous administration of L-NIL (3
mg/kg ip) with APAP (300 mg/kg ip) approximately doubled
ALT levels 4 h after APAP treatment in B6C3F1 mice. There-
fore, we also treated mice with L-NIL (10 mg/kg ip) 10 min
Fig. 5. Photographs of the liver sections for nitrotyrosine protein adducts after APAP in combination with vehicle, L-NIL, or
L-NAME. A: untreated controls showed no staining for nitrotyrosine in the liver. B: strong positive staining was seen in the
centrilobular sinusoids and centrilobular to midlobular hepatocytes 2 h after APAP. C: 6 h after APAP, a confluent staining for
nitrotyrosine was observed in the periportal hepatocytes. D: immunoreactivity with nitrotyrosine was expressed in the periportal
hepatocytes pretreated with L-NIL at 6 h after APAP. E: L-NAME exhibited intense expression of nitrotyrosine in the centrilobular
sinusoids 2 h after APAP. F: at 6 h after APAP, pretreatment with L-NAME caused intense staining for nitrotyrosine in the
centrilobular sinusoids. Original magnification, ⫻200 for A, D, and E and ⫻400 for B, C, and F.
Table 1. Effect of L-NIL or L-NAME on the serum concentration of nitrite/nitrate in mice treated with APAP
Control
Time After APAP Gavage
2h 6h
Vehicle
L-NIL L-NAME Vehicle L-NIL L-NAME
73.1⫾7.2 86.7⫾7.6 63.4⫾5.4 75.4⫾24.1 104.9⫾5.8 79.7⫾4.8 89.3⫾21.5
Data are means ⫾ SE (M) from 3–6 mice. There were no significant differences in nitric oxide (NO) concentrations among the treatment groups.
L-N
6
-(1-iminoethyl)-lysine (L-NIL) (10 mg/kg, ip) or N
G
-nitro-L-arginine methy ester (L-NAME) (10 mg/kg, ip) was administered 10 min before acetaminophen
(APAP) gavage.
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before APAP (300 mg/kg ip) or with L-NIL in combination
with APAP. As shown in RESULTS, the levels of ALT 6 h after
APAP were increased, and pretreatment with L-NIL slightly
reduced those, whereas cotreatment with L-NIL and APAP did
not significantly change them. Therefore, the discrepancy may
be due to the differences in the animal species, doses of
inhibitors, timing of their administration, or route of APAP
administration being used.
Since it has been suggested that L-NIL inhibits iNOS by
reacting with the protein at the enzyme active site, L-NIL does
not appear to suppress the expression of iNOS mRNA. On the
other hand, we demonstrated that L-NIL attenuated the expres-
sion of iNOS mRNA and iNOS protein in the liver. Although
the mechanisms by which L-NIL prevents iNOS expression in
this model remain unknown, Kang et al. (16) reported that, in
mouse peritoneal macrophages, L-NIL inhibited lipopolysac-
charide-induced activation of NF-B, which regulates iNOS
expression (37). Thus it may be plausible that L-NIL sup-
pressed iNOS mRNA expression in the liver treated with
APAP by inactivation of NF-B.
The bioactivation of APAP results in the formation of the
reactive metabolite, presumably N-acetyl-p-benzoquinone-
imine, which depletes intracellular sources of GSH (28). In the
current study, L-NIL suppressed the initial drop in hepatic total
GSH levels following APAP gavage, suggesting that the in-
hibitory effect of L-NIL on liver injury is partially mediated
through a decrease in bioactivation of APAP. The possibility
that L-NIL prevents APAP metabolic activation also suggests
that L-NIL could inhibit covalent binding to proteins of APAP.
Further studies will be required to elucidate the effect of L-NIL
on APAP covalent binding. The other possibility that L-NIL
maintained GSH levels is due to the reduced NO production in
the liver because reactive nitrogen species are scavenged by
GSH (18). Therefore, the mechanisms involved in the protec-
tive action of L-NIL against APAP-induced liver injury appear
to be multifactorial in this model.
The exogenous administration of NO donor (SNAP) exac-
erbated ALT release but did not affect the hepatic microcircu-
latory dysfunction following APAP gavage. These results sug-
gest that excess NO in the liver treated with APAP causes liver
injury and that NO may act directly on hepatocytes. Recently,
Liu et al. (22) reported that continuous administration of
O
2
-vinyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/
NO), a liver-selective NO donor, protected the liver against
APAP hepatotoxicity. The differences may be caused by dif-
ferent delivery of NO donor to the liver; V-PYRRO is me-
tabolized to NO preferentially in hepatocytes. Moreover,
excessive extracellular NO elicited by a bolus injection of
SNAP could mediate hepatocellular mitochondrial dysfunction
through its actions on an enzyme such as cytochrome c oxi-
dase, an enzyme constituting the distal end of the mitochon-
drial respiratory chain (4). The mitochondrial dysfunction
caused by SNAP could be amplified in addition to that by
APAP (14). In a preliminary study, we administered 10 mg/kg
of V-PYRRO/NO (Cayman) subcutaneously to mice gavaged
with APAP according to the experimental protocols by Liu et
al. (23). Since V-PYRRO/NO was supplied as a solution in
absolute ethanol, some animals received ethanol as vehicle.
Our preliminary study revealed that not only V-PYRRO but
also absolute ethanol as vehicle reduced ALT levels 6 h after
APAP gavage. Since a serum level of 5 mM ethanol has been
found to protect animals from APAP hepatotoxicity (35), the
effect of vehicle also could contribute to protective effect of
V-PYRRO/NO on APAP-induced liver injury, though we are
uncertain whether Liu et al. have used ethanol as a vehicle for
V-PYRRO/NO.
The toxicological effect of NO seems to occur through
peroxynitrite, a potent oxidant formed by reaction of NO with
superoxide. The finding of nitrotyrosine staining shown in the
hepatic CL sinusoids 2 h after APAP gavage, which is consis-
tent with those by others (12, 19), suggested that peroxynitrite
was formed early by the sinusoidal lining cells including KCs
and SECs. Thus it may be plausible that peroxynitrite injures
SECs in an early phase of APAP hepatotoxicity, resulting in
hemorrhage through the formation of gaps in their cytoplasm
(25, 36).
L-NIL diminished the initial nitrotyrosine formation in
the sinusoids, indicating that NO derived from iNOS in the
sinusoidal lining cells is involved in the formation of peroxyni-
Table 2. Effects of SNAP on serum ALT activity and on liver
microcirculation 6 h after APAP gavage
APAP Gavage
Vehicle, n⫽4 SNAP, n⫽3
ALT, IU/l 2,405⫾422 4,609⫾632*
No. of perfused sinusoids/field
Periportal 4.6⫾0.2 4.9⫾0.2
Centrilobular 3.8⫾0.3 3.9⫾0.3
No. of adherent leukocytes/100 m
Periportal 0.04⫾0.03 0.04⫾0.03
Centrilobular 0.04⫾0.04 0.00⫾0.00
No. of swollen SECs/field
Periportal 0.2⫾0.1 0.3⫾0.0
Centrilobular 0.2⫾0.1 0.3⫾0.0
Kupffer cell phagocytic activity
Periportal 1.0⫾0.1 0.5⫾0.1
Centrilobular 1.1⫾0.1 0.8⫾0.2
Area occupied by infiltrated erythrocytes,
m
2
/10 centrilobular regions
247⫾144 0⫾0
Data are means ⫾ SE. S-nitro-N-acetylpenicillamine (SNAP; 20 g/kg, ip)
or vehicle was administered 20 min before APAP (300 mg/kg) gavage. ALT,
alanine aminotransferase; SEC, sinusoidal endothelial cells. *P ⬍ 0.05 vs.
vehicle-treated mice.
Fig. 6. Effect of L-NIL on hepatic total glutathione (GSH) levels after APAP
gavage. The levels of hepatic GSH were determined in untreated controls and
1 and 2 h after oral gavage with 300 mg/kg APAP. Mice were pretreated with
L-NIL (10 mg/kg ip) 10 min before APAP. Data are means ⫾ SE. #P ⬍ 0.05
vs. untreated controls (time 0). *P ⬍ 0.05 for vehicle vs. L-NIL.
G65NO AND MICROVASCULAR INJURY
AJP-Gastrointest Liver Physiol • VOL 286 • JANUARY 2004 • www.ajpgi.org
trite. This finding is in agreement with that of Hinson et al. (11)
showing that aminoguanidine attenuated APAP-induced nitra-
tion of tyrosine in liver tissue. In considering the results of
microvascular inflammatory responses to APAP, KCs and
SECs appear to be sources of NO. However, iNOS protein in
the liver 2 h after APAP was not expressed in the sinusoidal
lining cells, suggesting that iNOS expression was not enough
to be detected by means of immunohistochemistry; even
mRNA for iNOS was marginally transcripted. Pretreatment
with L-NIL also minimized APAP-induced hepatic microcir-
culatory dysfunction, presumably by inhibiting KC function.
Because inactivation of KCs with gadolinium chloride restored
liver microcirculation in response to APAP (15), the inhibitory
effect of L-NIL on KC activity contributes to preserving he-
patic microcirculation, resulting in reduction of liver injury
(21, 27). However, the mechanisms by which L-NIL suppresses
KC function or whether KCs release NO in the early phase of
APAP hepatotoxicity remain unknown.
At later time point (6 h after APAP gavage), nitrotyrosine
staining was negative in the CL regions of the liver, whereas
iNOS staining was positive in the same regions. On the other
hand, nitrotyrosine staining was positive in the PP hepatocytes.
The results indicate that the location for nitrotyrosine staining
in the liver was shifted from the CL regions to the PP regions
with time after APAP treatment. The reduced intensity of
nitrotyrosine staining in CL hepatocytes 6 h after APAP may
have resulted from inability to nitrate protein because of
cellular lysis (31). On the other hand, the staining for nitroty-
rosine was observed in the PP hepatocytes with no evidence of
necrosis. These results are inconsistent with those reported by
others, who demonstrated nitrotyrosine expression in the CL
hepatocytes (19), but not in the PP ones (12), at 6 h after
APAP. The reasons for these differences remain unclear, al-
though it may be due to differences in animal species, response
to APAP, and/or differences in immunohistochemical proto-
cols. Our findings suggest that nitrotyrosine formation may
serve as a biomarker for the effects of reactive nitrogen species
and may not be relevant to the injury mechanism, because no
evidence of necrosis was noticed in the PP hepatocytes. In
addition, it may be speculated that peroxynitrite was reacted
with hepatic glutathione (endogenous scavengers of peroxyni-
trite), which are still reserved in the PP hepatocytes and
thereby protected against peroxynitrite-mediated oxidant stress
injury in these hepatocytes.
In contrast to L-NIL, L-NAME, a nonselective NOS inhibi-
tor, exacerbated APAP-induced liver injury. These findings are
consistent with the findings by others (11) showing that N-
monomethyl-L-arginine worsened the injury. The enhanced
liver injury was associated with aggravated APAP-induced
hepatic microvascular injury including KC phagocytic activity.
L-NAME caused upregulated expression of nitrotyrosine in the
hepatic CL sinusoids 2 and 6 h after APAP gavage, indicating
that peroxynitrite could lead to SEC injury. This possibility
was supported by the results of the present study showing that
the extrasinusoidal area occupied by infiltrated erythrocytes
was further increased by L-NAME. These results suggest that
NO-mediated nitration of cellular proteins in SECs at least in
part contributes to the progression of liver injury elicited by
APAP and that peroxynitrite is an important mediator of injury
to parenchymal cells (18) as well as SECs. However, as
mentioned above, whether the presence of nitrotyrosine-pro-
tein adducts is related to the injury remains to be elucidated.
The enhanced hemorrhagic necrosis by L-NAME also suggests
that endogenous NO derived from cNOS plays a critical role in
maintaining the functional integrity of SECs, because cNOS
appears to be localized exclusively to the SECs (32). cNOS has
been shown to associate with cytoskeletal proteins (1). Thus
inhibition of cNOS with L-NAME could result in disruption of
cytoskeleton, leading to changes in cell shape or to the forma-
tion of gaps. In addition, it has been reported that APAP
increased levels of hepatic lipid peroxidation in mice treated
with the iNOS inhibitor aminoguanidine (11) or in mice defi-
cient in iNOS (26). This suggests that exacerbated liver injury
elicited by APAP is mediated by lipid peroxidation during
NOS inhibition. However, Knight et al. (17) recently have
shown that there is no evidence of enhanced lipid peroxidation
in the liver treated with APAP in combination with aminogua-
nidine, indicating that lipid peroxidation is not involved in the
injury mechanism. Because of the antioxidant properties of
cNOS-derived NO, inhibition of cNOS could increase super-
oxide (9). Thus it is plausible that activated KCs generated
larger amounts of superoxide and thereby formed peroxynitrite
in the sinusoids. However, the mechanisms by which NO from
cNOS suppresses the activation of KCs remain unknown.
Recent studies reported that nonselective NOS inhibitors
reduced the sinusoidal perfusion and sinusoidal diameters dur-
ing endotoxemia (5, 30), indicating that cNOS inhibition
caused vasoconstriction and thereby augmented liver injury.
However, this is not seen in the current study, in which
L-NAME did not further reduce the sinusoidal blood flow as
well as sinusoidal diameter following APAP treatment. Thus
aggravation of APAP-induced liver injury by L-NAME is not
attributed to vasoconstriction.
In summary, selective inhibition of iNOS with L-NIL atten-
uated liver microcirculatory dysfunction elicited by APAP
gavage. In contrast, inhibition of cNOS by L-NAME potenti-
ated APAP-induced liver injury due to its effect on the liver
microcirculation including KC function. These results suggest
that NO derived from iNOS contributes to APAP-induced
parenchymal cell injury and hepatic microcirculatory distur-
bances, whereas NO-derived cNOS exerts a protective role in
liver microcirculation against APAP intoxication and prevents
enhanced liver injury.
GRANTS
This study was supported in part by National Institute on Alcohol Abuse
and Alcoholism Grant RO1-AA-12436.
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