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PII s0024-3205(97)01033-3
DIETARY
NUCLEOTIDES
HAVE CYTOPROTECTIVE
PROPERTIES
DAMAGED BY THIOACETAMIDE
M.I. Torres”.
‘Department
M.I. Fernandez’,
IN RAT LIVER
A. Gil’ and A. Rios’
of Cell Biology, Faculty of Sciences. University of Granada. Fuentenueva
Granada, Spain. ‘Department of Biochemistry and Molecular Biology.
s/n. 18071
(Received in final form October 13, 1997)
Summary
Liver cirrhosis has been induced with thioacetamide administered via different routes
in rats and other species. The oral intake of thioacetamide causes nodular liver cirrhosis
in rats characterized by extensive fibrosis occupying most of the hepatic parenchyma.
To characterize the cytological features of cirrhosis induced by thioacetamide, and the
degree of recovery obtained with dietary nucleotides, we made a morphometric study
of the hepatocytes in rats administered 300 mg/l of thioacetamide for 4 months, and in
rats receiving the same hepatotoxic treatment but allowed a 2-weeks recovery period
on a nucleotide-free
diet or a 250 mg/lOOg nucleotide-supplemented
diet.
Thioacetamide caused to cell damage and affected the ultrastructure of hepatocytes
leading to a decrease in cytoplasmic area together with increased nuclear and nucleolar
size. Dietary supplementation
with nucleotides
favoured recovery, restoring the
cytoplasmic (TN=491.7+9.6 vs TAA=305.1+3.7), nuclear (7X6+2.8 vs 97.4+2.9), and
nucleolar area of damaged hepatocytes
(5.6+0.3 vs 14.0+0.9). The injury from
thioacetamide intake increased liver collagen, but dietary nucleotides prevented hepatic
deposition
of this protein. This study supports
the hypothesis
that dietary
supplementation
with nucleotides is decisive in ensuring hepatocyte recovery after
thioacetamide-induced
liver damage, and that dietary nucleotides have antifibrotic
properties.
Key Words: hepatoeyte, thioacetamide,
liver cirrhosis, nucleotides, collagen
Thioacetamide (TAA) has been used in many studies of experimental toxicity to investigate liver function.
carcinogenesis and the cellular response to induced aggression.
Liver cirrhosis has been induced with
different doses of TAA administered via different routes in rats and other species (1-S). Many authors
have reported that TAA-induced cirrhosis is a suitable experimental model that gives rise to a disease
comparable to that occurring in humans (6,7). Despite the difference in the etiological agent, the
histological and biochemical changes in animals subjected to chronic exposure to TAA are similar to those
observed in human cirrhosis (6-S).
Regardless of the cause of disease, fibrogenesis in the cirrhotic livers is the result of a great increase in
collagen content. The consequent impairment in the exchange of macromolecules between hepatocytes and
plasma further exarcerbates the disease. Collagen synthesis is one of the main pathological events in liver
damage caused by hepatotoxic agents such as TAA. The synthesis of this protein, one of the major
components of the extracellular matrix, increases in chronic liver diseases including cirrhosis. We show
here that the deposition of excessive amounts of collagen is related with liver damage. The accumulation
of collagen is considered a marker of fibrosis; nonparenchymal cells (stellate cells) that subsequently
become myofibroblast-like
cells produce this extracellular matrix component during hepatic fihrogenesis
(9-11).
* To whom all correspondence
should be addressed
14
Nucleotide Cytoprotection in Cirrhosis
Vol. 62, No. 1, 1998
Thioacetamlde has been shown to cause ultrastructural changes that include nuclear and nucleolar
hypertrophy in hcpatocytes (12-14). Findmgs reported by Belyaev et al. (1.5) suggest that experimental
cirrhosis dcvclops as a result of a disruption in hepatocyte functioning, rather than necrosis of the
parcnchyma. Most cases of cirrhosts are believed to originate from a nonspecific reaction of the
hcpatocytcs to various cirrhogenic factors; this reaction involves the increased release of factors that
stimulate proliferation of the connective tissue. During cirrhosis, several interactions take place between
factors that stimulate and inhibit growth in nonparenchymatous
cells (15).
Protein-energy malnutrition is common in chrome liver disease, including cirrhosis, and can affect
both the structure and function of the liver (16,17). Liver cirrhosis requires adequate metabolic control
lo prevent complicalions and progression of the disease to an irreversible stage. An adequate supply of
energy. protein and other nutrients are essential for the regeneration of damaged hepatocytes in liver
cirrhosis (IX, 19) and contribute lo decrcasc morbidity m human patients (20,21). Recent studies have
suggested that dietark nucleotldcs are semi-essential nutrients. particularly in newborns and adults with
certain metabolic disorders, and are involved in tissue repair (22-25). Under these conditions dietary intake
of nuclcotidcs would spare the organism the energy cost of de novo synthesis; thus nucleotides salvage
may opllmize tissue function particularly during the rapid cell replication which accompanies recovery
from TAA injury (8.26). Extracellular nucleotides modulate hepatocyte growth (27) and regeneration (28).
After hepatic injury. the regeneration of new tissue is accomplished by accelerated synthesis of RNA and
DNA (29).
The present study was designed to delcrmine the effects of dietary supplementation
with
nucleotidcs on the process of liver tissue regeneration in rats with experimental cirrhosis induced by TAA.
Our findings illustrate the relationship between hepatic injury and the intake of TAA, and the effect of
nuclcotide supplementation.
Morphomctric studies were used to quantify the cytological characteristics
of cirrhosis and the effect of dietary supplementation with nucleotides on recovery of the damaged liver.
Material and methods
Female Wistar rats were used. All animals were housed and treated in accordance with the
recommendations of the American Physiological Society (30). The animals were divided into two groups:
one group was treated with 300 mg,0 TAA in drinking water ad lihitum during 4 months (n=21); the
control group (n=lS) was given water without TAA. Both groups were fed a standard chow diet during
the first 3 months and a semipurificd diet in the fourth month. The composition of the semipurified diet
is shown in Tdblc I.
Table
I. Semrpurified
control
diet composition
(Research
Department
of PULEVA,
Spain)
Composition
9
Chemist composition
%
Calcium casemate
Cellulose
Sugar
Corn starch
VKO
Minerals’
Vitamins’
225.5
50
150
446 27
100
24
0.129
Proteins
Carbohydrates
LIpIds
Minerals
Vitamins
DI-Methlonlne
Choline chloride
20
67.18
10
2.4
0.0129
03
0.11
Expressed
as Kg of dlet.VKO.
Mixture of olive oil (66%), soya oil
(23%) and medium
trlacllglycendes
from refined coconut oil (1 1%). ‘Mineral and vitamin levels as specified by IIAR
-
chain
(31).
After 1 months of treatment and 3 additional days wlthout TAA administration to eliminate its
acute effect, 5 control and 7 TAA-treated animals were killed to evaluate the alterations produced by
TAA. At this time treatment with TAA was stopped and the rest of animals in both the TAA and control
group wcrc divided inlo the following groups for recovery studies:
I) Control
group:
the
conrrol group was divided into two subgroups.
In one (control-control,
CC, n=S),
Vol. 62, No. 1,19!B
Nucleotide Cytoprotection in Cirrhosis
1.5
the animals received the semipurified diet during 2 weeks (8.26). The composition of this diet followed
the ILAR rules (31). The semipurified diet was essentially free of nucleotides, nucleosides, purine and
pyrimidines bases as assessed by high performance liquid chromatography
analysis. In the controlnucleotide group (CN, n=5) rats were given the semipurified diet supplemented with SO mg each of AMP.
IMP, GMP, CMP and UMP per 100 g feed during 2 weeks.
2) TAA group: this group was also divided into subgroups. Animals in the TAA-control subgroup (TC,
n=7) received the semipurified diet for 2 weeks. In the TAA-nucleotide
subgroup (TN, n=7) the
semipurified diet was supplemented with SO mg each of AMP, IMP, GMP, CMP and UMP per 100 g feed
during 2 weeks.
The average dietary intake during the TAA injury period was not significantly different in the
experimental and control group ([email protected] g/rat/day w 20.21+0.53 g/rat/day, respectively). Diets were
administered immediately after TAA treatment was stopped. During the recovery period the difference in
food intake between the subgroups fed the nucleotide-supplemented
diet did not reach significance
(20.03+0.09 g/rat/day in group TN ws 16.69+X51 g/rat/day in group CN).
Histological
studies
After the animals were killed, the liver was immediately removed and samples were taken from
the left lobe and processed for light and electron microscopy. For light microscopy the tissue was fixed
in 4% paraformaldehyde,
embedded in paraffin and examined for histological studies. For electron
microscopy samples were fixed in 3% glutaraldehyde in cacodylate buffer (O.lM. pH 73, postfixed in
1.5% osmium tetroxide and then dehydrated in acetone and embedded in Epon 812 resin. Ultrathin
sections were stained with uranyl acetate and lead citrate and observed in a Zeiss 902 transmission
electron microscope.
Measuremenr
of colfagen
Sections measuring 15 pm in thickness were deparaffinized and stained with Fast green and Sirius
red according to the procedure of L@z-de-L&n
et al. Absorbance of the eluted color was read in a
spectrophotometer.
Color equivalences were estimated according to the method described previously by
JimCnez et al. (32). The results were expressed by ,ug collagenimg protein.
Morphometric
analyses
From each liver sample, 10 randomly selected blocks were prepared for electron microscopy: three
of these were chosen for quantitative analysis. Prior to electron microscopy, semithin sections from each
sample were prepared, stained with toluidine blue, and used to facilitate the orientation of specimens with
respect to lobular architecture.
Tissue from each of animals was taken and cut it into small slices, and one section from each
ribbon was selected. In each animal from each group, 10 photographic negatives were obtained, each of
which showed 4 or 5 whole cells. The upper right comer of each field was photographed at xl 100. Three
blocks per fraction and experiment were randomly chosen, and from each block three ultrathin sections
were cut at equally spaced intervals not less than 200 pm.
Only whole cells in which the whole nucleus was visible were measured. The cytoplasmic. nuclear
and nucleolar areas corresponded to the areas of these profiles expressed as the number of pixels occupied
by the cytoplasm, nucleus and nucleolus. These parameters were measured with a Visilog 4.1.1 image
analysis program.
Statistical
analysis
All results were expressed
as the mean + SEM. A one-way analysis of variance (ANOVA) was
NucleotideCytoprotectionin Cirrhosis
16
Vol. 62, No. 1, 1998
used to compare TAA treatment 1’s the control group. A two-way ANOVA was used to evaluate the
combined cffec~\ of two variables (33). Comparison of the means was done using’ “a posteriori”
Bonferrom tests: p<O.OS was considered statistically significant. Homogeneity of variance was checked
in all cases with the Levenne test. If variance was heterogeneous, the Kruskal-Wallis nonparametric test
was used to compare the data. All data were cvaluatcd for statistical significance with BMDP software
(34).
Kesults
FeedIng Wlstar rats with TAA led to many morphological and structural changes in the liver. The
normal lobule structure had disappeared. and irregular and regenerative parenchymatous nodules were
delimited by fibrous scptac. which expanded to invade the parenchyma (Fig. IA). Injury resulting from
TAA was accompanlcd hy increased liver collagen deposition. The greast volume of collagen was
deposited in pcriccntral areas.
TAA also affected the ultrastructure of hepatocytes. Many nuclei became edematous and increase
m siLc until they occupied a large proportion of the cell volume, pushing other organelles into a narrow
layer of cytoplasm surrounding the nucleus. The nuclear envelope appeared electron-dense,
and the
nucleoli were enlarged and appeared granular and vacuolated. The cytoplasm contained few mitochondria
and a hypcrtrophic cndoplasmic reticulum. The rough cndoplasmic reticulum became disaggregated and
many of its cistcrnae were dispersed. dislocated and altered m length. Large numbers of free ribosomes
were seen in the cytoplasm. together with numerous lysosomcs and some lipid droplets (Fig. 2A)
Atier the recovery pcrlod. the livers 01 animals that consumed a diet supplemented with
nucleotidcs during 2 weeks after trcatmcnt with TAA (TN group) showed more marked histological
recovery than group TC (Fig. IB, IC).
Lghl
mlcrogrdphs of
prolitkratlon
A) TAA group: B) TN group: c‘) TC group. +
of bile ducts: arrow= tlbrous scplac. (OrigInal
regions ol l’ibrosls and
magnification
X 80).
Vol. 62, No. 1, 1998
Nucleotide Cytoprotection in Cirrhosis
17
In group TN there were fewer fibrous septae and an increase in the surface occupied by normal
hepatic parenchyma. At the ultrastructural level the shape and size of the nucleus and nucleoli were
normal. and the cytoplasm contained normal-appear ing mitochondria and rough endoplasmic reticulum
(Fig. 2B). In group TC the nucleus showed signs of damage, and the nucleoli were enlarged and
vacuolated. As the group TAA. the hepatocytes also showed many alterations, such as the accumulation
of lipid droplets and changes in mitochondria and in the rough endoplasmic reticulum. However, in this
group we observed some signs of recovery in responr ;e to withdrawal of the hepatotoxic agent (Fig. 2C).
Fig. 2
(A) Electron micrograph of liver tissue in thioacetamide-induced
cirrhosis (TAA group).
Hepatocyte with enlarged nucleus, vacuolated nucleolus (head arrow), and alterations in the
mitochondria
and endoplasmic
reticulum.
(B) In group TN, the nucleus and nucleolus have
returned to normal size and appearance,
and alterations in the cytoplasm are no longer seen.
(C) In group TC, a degree of nuclear and nucleolar hypertrophy is still evident, and alterations
in the cytoplasm similar to those in group TAA can still be seen. N= hepatocyte nucleus.
(Original magnification
X 1,650).
18
Nucleotide Cytoprotection in Cirrhosis
Vol. 62, No. 1, 1998
In conW rats fed the nuclcotide-supplemented
diet for 2 weeks (group CN) we observed large
amounts of rough endoplasmlc reticulum in the hepatocyte cytoplasm (Fig. 3).
Fig.
Elcclron
micrograph
hcpalocytes
ol group
are ohservcd.
CN.
3
large amounlb
N= hepatocylc
nuclcu~
ol’ rough endoplaamlc
rckulum
(Original
X 2,200).
magnification
( I) in
the
In rats given TAA WC not& a significant incrcasc m the amount of collagen in the liver (Table
2) in comparison with the control group. After the recovery period, collagen deposits in rats given
nuclcotides had decreased significantly in comparison with group TAA and group TC. which was given
a scmipurificd dicl during recovery (Table 2). These findings suggest that dietary supplementation with
nuclcolidcs dccrcascd the dcgrce of fibrogcncsls induced by TAA.
Table 2 Hepatlc collagen content I” group control, TAA and the other expenmental
groups given a diet
supplemented
with nucleotides TN) or a semipurified control diet (TC) after wlthdrawal of TAA and their
respective control groups (CN, CC)
Group
n
pg collagen/mg
protein
Control
TAA
Experimental
CN
cc
TN
TC
5
7
89.40+2.15
121 9255 84”’
5
5
7
7
89.582 2 50
92.50+1 40
101.64+_3.36tec2
1 14.03+_4.i4c’~2
Groups
Results are expressed as mean + SEM; n= number of animals in each group. a slgnlflcance vs control
and their control groups:
group, b significance vs TAA group: ‘, significance between TAA-experimental
d signlflcance between TN and TC groups. ’ P<O 001: ’ P-&01; 3:P&O5
Nodular clrrhosls induced by the oral admmlstration of TAA led to a significant dccrcasc in
cytoplasmic area in hepatoyctcs
(Table 3). After the recovery period, the TM-experimental
groups TN
and TC showed some degree of ultrastructural Improvement, as evidenced by the differences in
cytoplasmic arca with respect to group TAA. The values in animals given dietary nucleotide supplements
(TN) wcrc closer to those in their respective control group than to the areas in group TC. Hepatic recovery
was influcnccd both by the withdrawal of TAA (F=336.41; p<O.oOl) and by dietary supplementation with
nuclcorides (F= 160. IO: p=<O.OO
I ).
Vol. 62, No. 1, 1998
Nucleotide Cytoprotection in Cirrhosis
19
Table 3 summarizes the findings for nuclear area in the different groups. The oral administration
of TAA significantly increased the area of the hepatocyte nucleus. After 2 weeks of recovery the values
had begun to return to normal in all groups, and the nuclei appeared less hypertrophic than at the start
of recovery. Nuclear area, like cytoplasmic area, approached control values in group TN. Analysis of
variance showed that the type of diet had a significant influence on nuclear area in cirrhotic animals
(F=10.04; p=O.oOl).
The nucleus/cytoplasm
index (N/C) was greatest in rats given TAA for 4 months (Table 3). This
reflected the decrease in cytoplasmic area and the increase in nuclear area caused by TAA. After 2 weeks
of recovery the N/C index had begun to normalize. As found for cytoplasmic and nuclear area, the greatest
recovery was seen in rats given dietary nucleotides. Treatment with TAA increased nucleolar area. After
recovery, this parameter had decreased in all experimental groups (Table 3). The value in group TN and
its corresponding control group (CN) were more similar to each other than were the values in rats fed the
nucleotide-free diet (TC) and the corresponding control group (CC). After feeding with the semipurified
control diet (TC), nucleolar area remained significantly greater than in the corresponding control animals.
In cirrhotic rats the type of diet accounted for most of the difference (F=44.63; p<O.OOl).
Table 3. Mean values for nuclear, cytoplasmic and nucleolar area, and nucleus/cytoplasm
index (N/C),
in rats treated with thioacetamide
(TAA), controls, and animals recovered with the control
(TC) or
nucleotide-supplemented
(TN) diet for 2 weeks.
Group
n
Cytoplasmic
TAA
7
305.1+3,7”
C
5
TN
Area
Nuclear
N/C Index
Nucleolar
97.4*2,9=
0.23~0.01”
14.0+0.9”
515.3+_10.7
83.522.2
0.15~0.00
6.4kO.3
7
491.7+9.6b’2
73.6k2.8
0.14+0.00~‘”
5.6kO.3”
TC
7
261 .3+_4.6b”‘d’
69.4+2.0b’=’
0.21 +O.Ol “c’d’
8.9+0.7’-’
CN
5
534.0+9.5
86.8~2.6
0 15+0.01
6.350.3
cc
5
518.5+_12.4
88.9+2.6
0.1 S+O.OO
6.1~0.4
Area
W”
Area
-
Results are expressed as mean (elm* )+ SEM. n= number of animals. ‘: significance vs control group;
! significance vs TAA group; ‘: significance between T&Y-experimental
groups and their corresponding
control groups after recovery period: ‘: signification vs TN2 group. ‘: p<O.OOl ; ‘: pcO.001; ‘: p&05.
Discussion
Treatment with TAA administered orally at doses and for periods similar to those we used in the
present study is known to cause cirrhosis-like hepatic lesions characterized by histological and metabolic
features that closely parallel the alterations in human cirrhosis of different origins (6,8,). When hepatic
lesions are advanced, histological and functional recovery of the liver is unlikely. Recovery from nodular
cirrhosis may occur when the hepatotoxic agent is withdrawn, the speed of recovery depending on the
animal’s nutritional status. The degree of structural recovery depends on several factors, among which
dietary factors are of great importance (16,35-37).
Quantitative findings for collagen demostrated that in TAA-induced
hepatic cirrhosis, the
deposition of hepatic collagen was greater than in control animals. Therapy for hepatic fibrosis should
therefore interfere specifically with the production of hepatic connective tissue proteins. In the present
study, dietary nucleotides decreased the hepatic deposition of collagen. Hepatic collagen production has
been reported to be disminished when intracellular levels of CAMP are high (38). So, we hypothesize that
dietary nucleotides might modulate hepatic collagen production by increasing CAMP levels.
20
Nucleotide Cytoprotection in Cirrhosis
Thtoacctamtdc
tn mean cell size and
specimens) occurs in
enlargement of nuclear
nuclear surface arca.
Vol. 62, No. 1, 1998
reduces cytoplasmatlc area and increases nuclear and nucleolar size. The reduction
incrcasc in the N/C index (which appears as nuclear crowding in histological
hepatocarcinoma
(39). The increase in N/C index was caused mainly by the
size. Treatment with TAA results in nuclear swelling, which implies increased
Thloacetamtdc has a vartcty of effects on nuclear metabolism in the liver (40.41). Evidently.
damage to the nucleus results in important changes in its physical properties. Nuclear size in hepatocytes
is probably rclatcd with DNA content. Thioacetamide Increases the size of nucleoli; this may represent
a specific cffcct on the metabolism of nuclcoproteins in the liver parenchyma (42-44).
In control rats fed the nuclcottdc-supplemented
diet for 2 weeks, the hepatocytes contained larger
amounts of rough cndoplasmic reticulum.
Diets depleted in nucleotides may affect hepatic nucleotide
metabolism and RNA synthesis and support the hypothesis that liver nucleotide metabolism is modulated
by the availability of dietary nuclcotides (45.46).
Dietary nuclcottdcs modulate hcpatocytc growth and liver regeneration.
Ogoshi et al. (47,4X)
showed that nucleotides admimstcrcd
parenterally improved liver functioning and accelerated the
rcstoratton of nitrogen balance after liver damage or partial hepatectomy. We have previously shown that
dietary supplcmcntation with nucleotidcs influenced the percentage of binucleated hepatocytes, suggesting
that nucleotidcs affect regulation of the mitotic cycle and thus influence cell growth (26). Ohyanagi et al.
(27) have shown that nuclcotides enhance the growth of cultured hepatocytes. These studies suggest that
cxogcnously administcrcd nucleotidcs affect hcpatic composition and function. Dietary nucleotides may
thcrcforc hc especially important in meeting nucleotide needs when the liver’s capacity to supply
pcrl’ormcd nuclcotidcs is dimimshed by discasc or in,jury.
Our results show that dietary nuclcottdcs have cytoprotective properties in liver cirrhosis induced
by TAA. However. their mechanisms of acllon are still not fully clear. The antifibrotic effect 01
nuclcottdcs
may be associated with protection of hcpatocytes
from damage, attenuation of the
inflammatory reaction. and interfcrencc with collagen metabolism. We have used evidence from statistical
analysts of morphomctric and quantitative data. together with descriptive observations, to compare the
cffccts of a scmipurified control diet free of nucleotides. and a diet supplemented with nucleotides in
animals that had been made cirrhotic with TAA. The effect of dietary supplementation with nucleotides
in reducing liver tissue damage by nodular cirrhosis was greater than the effect of withdrawal of TAA.
WC therefore conclude that nuclcotidcs may bc effective in the treatment of cirrhosis, and that a
nuclcotide-free diet may compromises rccovcry from TAA-induced damage in the liver.
Acknowledgements
Thts study was supported by the Project SAL90-0615 CICYT. Puleva and Abott laboratories,
Granada, Spain
WC thank Ms. Karen Shashok for translating the original manuscript into English.
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