EL-MINIA MED., BULL., VOL. 18, NO. 2, JUNE, 2007
Ali & Taha
THE PROTECTIVE ROLE OF ALLOPURINOL AGAINST
PARACETAMOL INDUCED HEPATOTOXICITY RESULTING FROM
PEROXINITRITE FORMATION.
HISTOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY
By
Azza Hussein Ali* and Hanan Ali Taha**
Departments of Histology, *El-Minia Faculty of Medicine
and **Internal Medicine, Beny-Suef Faculty of MedicineABSTRACT:
Paracetamol is an effective and safe pain-relieving drug when therapeutic
doses are taken. Vascular injury and accumulation of red blood cells in the space of
Disse (hemorrhage) is a characteristic feature of paracetamol hepatotoxicity, another
postulated mechanism of paracetamol injury is peroxinitrite formation. Therefore, the
objective was to investigate if intracellular events in hepatocytes and endothelial cells
are responsible for the cell damage. A total of 30 adult male albino rats were
randomized into 3 groups. Apart from the routine feeding regimens, group 1 was
used as control group; group 2 animals received an intraperitoneal injection of 300
mg/kg paracetamol. Groups 3 received 100 mg/kg zyloric or 20 ml/kg water p.o. 18 h
and 1 h before paracetamol administration. Animals were killed after 1 and 6 hours
respectively after paracetamol treatment. Blood samples were collected for detection
of hemoglobin and alanine transferase (ALT) activities. Paraffin sections of liver were
prepared and stained with H & E. Nitrotyrosine staining was assessed by
immunohistochemistry. Paracetamol treatment caused vascular nitrotyrosine staining
within 1 h. vascular injury (hemorrhage) occurred between 2 and 4 h. This paralleled
the time course of parenchymal cell injury as shown by the increase in plasma alanine
aminotransferase activities. Treatment with zyloric (100 mg/kg), which prevented
mitochondrial injury in hepatocytes, strongly attenuated vascular nitrotyrosine
staining and injury. This protective effect of zyloric treatment suggests that,
peroxynitrite formation in sinusoidal endothelial cells and hepatocytes may be critical
for vascular injury after acetaminophen overdose.
KEY WORDS:
Hepatocytes
Zyloric
Paracetamol
Histology
Protective
Nitrotyrosine
isoenzymes, is essential for the development of paracetamol-induced liver
toxicity2. NAPQI is readily conjugated
with glutathione and excreted from
hepatocytes3. However, excessive
NAPQI formation results in covalent
binding to sulfhydryl groups of
proteins2,4. Although protein binding is
a critical early event in paracetamol
hepatotoxicity, this mechanism alone
cannot explain the severe cell injury.
INTRODUCTION:
Paracetamol is a well known
anti-inflammatory drug. However, an
overdose of it causes centrilobular
necrosis, which in severe cases can
lead to liver failure, in both experimental animals and humans1,2. It is
well established that formation of a
reactive metabolite, presumably Nacetyl-p-benzoquinone
imine
(NAPQI), by microsomal P450
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Ali & Taha
overdose20,21. Therefore, the objective
of this investigation was to test the
hypothesis that peroxynitrite plays a
critical role in the mechanism of
paracetamol-induced
hepatocellular
toxicity and the possiple protective role
of allopurinol on such toxicity.
Therefore, several amplifying mechanisms have been postulated. Paracetamol treatment leads to Kupffer cell
activation5 and recruitment of neutronphils into the liver6. Furthermore, paracetamol metabolism causes mitochondrial dysfunction7-9, which results in
mitochondrial oxidant stress10 and
peroxynitrite formation11. Recently, it
could be shown that selective scavenging of peroxynitrite with glutathione
(GSH) effectively protects parenchymal cells in the liver against
paracetamol-induced cell injury despite
continued
mitochondrial
oxidant
stress12. This suggests that peroxynitrite plays a critical role in the
mechanism of paracetamol-induced
hepatocellular toxicity. Microvascular
disturbances and injury may also be
relevant for the progression of paracetamol-induced liver injury. Walker et
al., described sinusoidal endothelial
cell (SEC) injury with trapping of red
blood cells in the space of Disse
(hemorrhage) during paracetamolinduced liver injury in mice13,14.
Recently, Ito et al., demon-strated SEC
swelling and impaired endothelial
scavenger function, which preceded
parenchymal cell injury15. Subsequent
accumulation of erythro-cytes in the
space of Disse and reduced sinusoidal
blood flow indicate sub-stantial microvascular dysfunc-tion15. These microvascular changes may cause ischemic
injury, platelet aggre-gation and
thrombosis, neutrophil accumulation
and inflammatory injury16. Severe
hemorrhage can cause hypovolemic
shock17. However, the mechanism and
pathophysiological relevance of these
events for paraceta-mol-induced liver
injury remain unclear. Activated
Kupffer cells can generate reactive
oxygen and nitric oxide18 and may
cause vascular injury19. Kupffer cells
have been implicated in the mechanism
of hepatocellular injury and peroxynitrite formation after paracetamol
MATERIALS AND METHODS:
Animals: Thirty male Swiss Albino
mice weighing 17-20g were obtained
from the university animal house and
housed
in
an
environmentally
controlled room with 12 h light/dark
cycle. They were acclimatized for one
week. Food and water were supplied
ad libitum. All animals were fasted
overnight before the experiments
Animals were allocated in three groups
of ten animals each. Group one was
used as control group,received neither
poaracetamol nor drug treatment
.Group two animals received an
intraperitoneal injection of 300 mg/kg
paracetamol (Sigma Chemical Co., St.
Louis, MO), dissolved in warm saline
(15 mg/ml)].Group three animals
received 100 mg/kg zyloric or 20
ml/kg water p.o. 18 h and 1 h before
administration10,11. At selected times
after paracetamol treatment; animals
were killed by cervical dislocation.
Blood was drawn from the vena cava
into
heparinized
syringes
and
centrifuged. The plasma was used for
determination of alanine aminotransferase (ALT) activities (Test Kit DG
159-UV (Sigma Chem. Co., St. Louis,
MO) and expressed as IU/liter.
Immediately after collecting the blood,
the livers were excised and rinsed in
saline. A section from each liver was
placed in 10% phosphate buffered
formalin to be used in histochemical
analyses. A portion of the remaining
liver was frozen in liquid nitrogen and
stored at -80 degrees C for later
hemoglobin determination as described
in detail6.
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Ali & Taha
Bonferroni t test. If the data were not
normally distributed, we used the
Kruskal-Wallis Test (nonparametric
ANOVA) followed by Dunn's Multiple
Comparisons Test. P < 0.05 was
considered significant.
Histology and Immunohistochemistry.
Formalin-fixed tissue samples
were embedded in paraffin, and 5-µm
sections were cut. Replicate sections
were stained with H&E for evaluation
of necrosis (35). All sections were
obtained from the left lateral lobe.
Preliminary studies using several livers
showed no difference in necrosis or
nitrotyrosine staining between the
different lobes of the liver in this
model. Nitrotyrosine staining was
assessed by immunohistochemistry
with the DAKO LSAB peroxidase kit
(K684; DAKO, Carpinteria, CA),
which was used according to the
manufacturer's instructions. The antinitrotyrosine antibody was obtained
from Molecular Probes (Eugene, OR).
RESULTS:
Administration of 300 mg|kg
paracetamol had no effect on liver
tissue at 1 hour but caused severe
centrilobular necrosis with haemorrage
at 6 hours (fig 1). Based on the release
of ALT, cell damage was first evident
at 2 hours (table). The sinusoids
showed haemorrage and the time
course of Hb accumulation assures that
sinusoidal injury occurred between 2-4
hours after paracetamol administration
(table). So, the damage to the endothelial cells was parallel to hepatocyte
injury.
Nitration of BSA in vitro.
The nitration of proteins by
peroxynitrite (Upstate Biotechnology,
Lake Placid, NY) was determined as
the intensely yellow phenolate of
nitrotyrosine. Briefly, BSA (Sigma
Chem. Co., St. Louis, MO) was added
to 60 mM carbonate buffer (pH 9.6)
with a final concentration of 2 mg/ml.
Varying concentrations (50 µM, 200
µM, 500 µM, 1 mM) of allopurinol or
N-acetylcysteine were added to the
BSA-carbonate buffer. Peroxynitrite
was then added to each solution (final
concentration of 1.40 mM) to nitrate
BSA.
Immunohistochemical staining for
nitrotyrosine demonstratesd selective
staining of endothelial cells at 1 hour
after paracetamol administratio. On the
other hand, staining of centrilobular
hepatocytes was evident at 6 hours (fig
2). Pretreatment with zyloric could
protect liver parenchyma from paracetamol toxicity. It prevents hepatocyte
and endothelial cell necrosis (fig 3)
and prevents the increase in plasma
ALT values (table). Also, zyloric treatment prevented haemorrhage, which
means healthy sinusoidal endo-thelial
cells (table). Consequently, zyloric
treatment prevented hepatocyte and
endothelial cell nitrotyrosine staining
(fig 4).
Statistics:
All results were expressed as
mean – SE. Comparisons between
multiple groups were performed with
one-way ANOVA followed by
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Ali & Taha
Fig. 1: photomicrographs of rat liver sections stained with haematoxilin and
eosin, showing A) norml hepatic architecture. B) 1 hour after paracetamol
administration showing minimal evidence of necrosis (star). C) 6 hours after
paracetamol administration showing confluent areas of necrosis around the
centrilobular regions. ( x 200)
Fig. 2: Immunohistochemistry of rat liver sections for nitrogtyrosine NT protein
adducts. A) No evidence for NT staining in the control group. B) 1 hour after
paracetamol administration. No NT staining was localized in vascular endothelial
cells. C) 6 hours after paracetamol administration showing confluent centrilobular
NT staining in hepatocytes (arrow). ( A, B x 200 & C x 400).
Fig. 3: A photomicrograph of rat liver sections stained with haematoxilin and
eosin, A) 1 hour after paracetamol administration showing confluent areas of
necrosis around the centrilobular region (arrow). B) 6 hours after paracetamol
administration (pretreated with zyloric), the liver was histologically normal. (A
x 400 & B x 200).
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EL-MINIA MED., BULL., VOL. 18, NO. 2, JUNE, 2007
Ali & Taha
Fig. 4: Immunohistochemistry of a rat liver section for NT protein adducts. A)1
hour after zyloric- paracetamol administration, the liver was histologically
normal with very limited NT staining in sinusoidal endothelial cells. B) 6 hours
after zyloric- paracetamol administration, the liver was histologically normal
with no evidence of NT staining.
16000
14000
12000
10000
ALT
8000
7000
8100
15200
12000
6000
60
4000
60
0
2000
2
4
6
1900
60
0
1
2
3
4
Time (h)
Table 1: Time course of liver injury (plasma ALT activities) and haemorrhage (liver
Hb content) after administration of paracetamole. Values were given as percentage of
baseline (ALT: -8U/ L; Hb:0.4 – 0.1 mg/g protein). Data represent means – SE of n=
10 animals per time point.(cmpared to untreated control).
4500
4000
3500
ALT values
3000
2500
2000
1500
1000
500
0
1
2
3
Animal Groups
Table 2: Effect of zyloric on paracetamole – induced liver injury (plasma Alt activities).
Data represent means – SE of n = 10 animals per time point.
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EL-MINIA MED., BULL., VOL. 18, NO. 2, JUNE, 2007
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periportal areas25,26. Activation of these
cells results in a predominantly periportal to midzonal injury27. In contrast,
paracetamol causes a strict centrilobular necrosis and hemorrhage with the
earliest and most severe injury
affecting the cells closest to the central
vein11,22. Thus, overall results are
consistent with a number of observations, which do not support a role of
Kupffer cells in vascular peroxynitrite
formation and injury.
DISCUSSION:
Overdose of the widely used
analgesic drug paracetamol causes
hepatotoxicity, which can in severe
cases lead to liver failure in experimental animals and humans1. There is
increasing evidence that supports a
critical role of peroxynitrite in the
pathophysiology paracetamol-induced
liver cell injury. Peroxynitrite is a
nitrating agent and a potent oxidant,
which can cause oxidative damage to
all types of cellular macromolecules.
Peroxynitrite is generated by the
spontaneous, reaction of nitric oxide
and superoxide14. It is an aggressive
oxidant, which can cause nitration of
proteins (e.g., nitrotyrosine formation)
and induce oxidative damage to all
types of cellular macromolecules.
Increased levels of plasma nitrite and
nitrotyrosine formation indicated that
nitric
oxide
and
peroxynitrite,
respectively, are indeed formed during
acetaminophin
hepatotoxicity11.
Previous studies suggested that
Kupffer cells are activated after AAP
overdose5 and are relevant contributors
to the overall liver injury in rats20.
More recently, similar results was
reached using the mouse model21.On
the contrary,other studies mentioned
that kupffer cells play at most a minor
role
in
the
pathophysiology.
Gadolinium chloride (GdCl3), which
functionally inactivates Kupffer cells
to produce less reactive oxygen23,
neither reduced the early vascular nor
the later parenchymal cell staining for
nitrotyrosine. Moreover, GdCl3 treatment had no significant effect on
vascular cell injury (hemorrhage) or
hepatocellular necrosis. These data
consistent with recent data showing no
attenuation of nitrotyrosine staining or
injury in phox-deficient mice, which
have no functional NADPH oxidase,
the main superoxide producing enzyme
in Kupffer cells24. In addition, the most
active Kupffer cells are located in the
Other vascular sources of
reactive oxygen formation, e.g., neutrophils, can be excluded, because
these cells accumulate at later time
points and may be relevant for later
hepatocellular events. Although neutrophils accumulate in the hepatic vasculature during AAP-induced liver
injury, antibodies against 2 integrins,
which prevent a neutrophil-derived
oxidant stress, did not attenuate AAPinduced liver injury7 These data
suggest that neither Kupffer cells nor
neutrophils are the main source of
superoxide
and
peroxynitrite
formation.
In parenchymal cells, AAP
induces mitochondrial swelling28 and
dysfunction7,8, oxidant stress10, cytochrome c release9, peroxynitrite formation11 and a reduction in cellular ATP
levels10. Preventing mitochondrial
dysfunction with allopurinol treatment
eliminated the oxidant stress, peroxynitrite formation and cell injury10,11. On
the other hand, if peroxynitrite was
scavenged by GSH, injury was
attenuated despite continued mitochondrial dysfunction12. The findings
suggest that peroxynitrite is a critical
mediator of AAP-induced liver injury.
Our present data show that allpurinol
treatment prevented the vascular
nitrotyrosine staining and hemorrhage.
It was previously shown that AAP
caused severe depletion of GSH and
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EL-MINIA MED., BULL., VOL. 18, NO. 2, JUNE, 2007
injury in cultured sinusoidal endothelial cells29. Further support for the
relevance of sinusoidal peroxynitrite
generation was also provided by the
observation that the overexpression of
vascular
glutathione
peroxidase
reduced liver injury and prolonged
survival after acetaminophen overdose.
Glutathione peroxidase can not only
metabolize peroxides but is also an
effective reductase for peroxynitrite10.
Thus, vascular peroxynitrite formation
occurs early in the pathophysiology of
acetaminophen-induced liver injury
and may be relevant for later hepatocellular events. These observations
document the capacity of sinusoidal
endothelial cells to metabolically
activate AAP. Since NAPQI, the
reactive metabolite of AAP, is
responsible for the mitochondrial
oxidant stress and peroxynitrite
formation in parenchymal cells11, and
the fact that allopurinol prevented
vascular nitrotyrosine staining and
injury suggests that paracetamol may
have caused a mitochondrial oxidant
stress and peroxynitrite formation in
endothelial cells. Thus, endothelial cell
damage and hemorrhage occurred
parallel to the parenchymal cell injury
through similar mechanisms.Without
interventions, the massive hemorrhage
can lead to hypovolemic shock and
death, as shown in other models of
sinusoidal endothelial cell injury17.
However, the early vascular injury
between 2 and 4 h had no effect on
hepatic ATP levels10. These results
suggest that the initial hemorrhage
does not lead to significant tissue
ischemia.
Ali & Taha
vasculature but play an important role
in clearing a large number of
macromolecules and colloids from the
circulation. Collagen-, mannose-, Fc
gamma-, and hyaluranon scavenger
receptors are vital for the turnover of
extracellular matrix proteins and the
removal of immune complexes30.
Reduced uptake of formaldehydetreated serum albumin as early as 2 h
after AAP administration demonstrated
dysfunction of the hyaluranon scavenger receptor15. However, it is unclear
if peroxynitrite is generated in hepatocytes or in the vasculature. Vascular
nitrotyrosine staining was evident
before liver injury between 0.5 and 2 h
after acetaminophen treatment. However, liver injury developed parallel to
hepatocellular nitrotyrosine staining
between 2 and 6 h after acetaminophen. A high dose of allopurinol
(100 mg/kg) strongly attenuated
acetaminophen protein-adduct formation and prevented the mitochondrial oxidant stress and liver injury
after acetaminophen..
In summary, our investigation
demonstrated an initial nitrotyrosine
staining of sinusoidal endothelial cells
of the centrilobular areas, which
indicates that peroxynitrite formation
begins in the sinusoids during the early
phase after acetaminophen administration. The subsequent intracellular
peroxynitrite generation in hepatocytes
increased in parallel In addition to the
initial vascular NT staining, our data
showed a progressive intracellular
staining in hepatocytes. Based on the
magnitude of NT staining in
hepatocytes, it appears unlikely that
vascular peroxynitrite could be
responsible for the intracellular NT
adduct formation in hepatocytes. Thus,
peroxynitrite must have been generated
in hepatocytes. with parenchymal cell
injury.
Nevertheless, injury and prolonged dysfunction of sinusoidal
endothelial cells, even without severe
hemorrhage, can be expected to have a
negative impact on liver function.
Sinusoidal endothelial cells have not
only a barrier function in the liver
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EL-MINIA MED., BULL., VOL. 18, NO. 2, JUNE, 2007
Further studies are necessary to
investigate the unclear downstream
mechanisms of acetaminophin toxicity
after peroxynitrite formation. This
indicates that there is a critical window
where peroxynitrite needs to be
eliminated to attenuate cell injury. To
conclude, it is recommended to
investigate in a clinical trial the use of
allopurinol as a treatment in cases of
drug induced hepatotoxicity.
Ali & Taha
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‫الدور الوقائى لأللويوورووو ضد السموة الكيدوة التى وحدثها الياراسوتامو‬
‫والواتجة عن تكون مادة اليوروكسوووترات‪ .‬دراسه هستولوجوه وهستوكوموائوه‪.‬‬
‫عزة حسون على ‪ -‬حوان على طه‬
‫قسم الهستولوجي‪ -‬كلوة طب المووا‬
‫وقسم الياطوه‪ -‬كلوة طب يوى سووف‬
‫يعتبررعقارررلعقرابلعر رري تلمولقمرراقرام رر شلئقةررلسعخقرم ررتانرتقرمقراقتعلاي ر قب عاررلئق‬
‫متزرينهقا قتلثيعق لتقالىقرا بنقوهوقملقر تهنفت قهذهقرانعر ر قوقرنقر رتانمئقثةثرخقم موارلئق‬
‫ماقرا عذراقرابيضلءققت وشئقرام مواخقرمواىقراضلبا قماققسعراقصحيحخقرابشي قغيعقمعلا ر ق‬
‫واوملئقرام موا قراثلشي قبعرلعقرا بلعر يتلمولقرملقراثلاثر قفررنقاوا رئقبلازيلوعيرلققبرلقتعرلاىق‬
‫رابلعر يتلمولق قوتتقذبحقرافسعراقوراذئقايشلئقماقرانتقاعملقرشزيملئقرا بنقوش بخراهيمو لوبياق‬
‫ثتقراذئقايشلئقماقرا بنقالنعر قراه تواو ي قوانعر خقرا يميلءقرامشلاي ق(شيتعوتيعوزيا) قوقنق‬
‫اوحظقراقتعلاىقرابلعر يتلمولقرنىقراىقاللق بيعقفرىققوظرلس قرا برنقفررنقرعتفرزقرشرزيتقرممشرياق‬
‫تعرش فيعيزقو ذالقش بخقراهيمو لوبياقبلانتقوبفحصقرا بنقم هعيلقتبياقحنوثقتل قورضرحقفرىق‬
‫رااةيلقحولقراوعينقرامع زىورحترلاقفىقرا يوبقرانموير قوشزير قفرىقت رلوي قنمق مرلق ر لئق‬
‫رعتفلالقري لبيلقورضحلقالشيتعوتيعوزياقوراذىقيعزىقراي قرمضعرعقراشل مر قبلا برن قوذارلقاةفرلق‬
‫الم موا قراضلبا قو رذالقرام موار قرامعلا ر قبرلمالوبيوعيشولقحيرثقبرنئقاةيرلقرا برنق رليم ق‬
‫و ذالقش بخقرمشزيتقوراهيمو لوبياقق ملق لاقراتفلالق لبيلقالشيتعوتيعوزياقمملقمملقيؤ رنقرارنوعق‬
‫راوقلسىقاألاوبيوعيشول ققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققققق‬
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