Egypt. J. Exp. Biol. (Zool.), 3: 91 – 100 (2007)
© The Egyptian Society of Experimental Biology
RESEARCH ARTICLE
Saber A. Sakr
Hany A. Abd-El Samie
Rania A. Sheir
EFFECT OF CHROMIUM ON CARBON TETRACHLORIDE-INDUCED
HEPATOTOXICITY IN MICE: HISTOLOGICAL AND HISTOCHEMICAL STUDIES
ABSTRACT
Carbon tetrachloride (CCl 4 ) is a hydrocarbon
compound that widely used in many
industries. Intraperitoneal administration of
carbon tetrachloride at dose level of 0.01 ml /
g bw 3 days / week for 5, 7, and 9 weeks
induced marked histological, histochemical
changes in liver of albino mice. The
histological changes included congestion of
blood
vessels,
leucocytic
infiltration,
cytoplasmic vacuolation, fatty infiltration,
necrosis, fibrosis and finally cirrhosis. CCl 4
also caused significant reduction in hepatic
polysaccharides and total protein contents.
Treating mice with CCl 4 caused significant
increases in serum ALT, AST and total lipids
while it induced significant decrease in serum
albumin. These alterations were timedependent. Treating animals with both carbon
tetrachloride and chromium (Cr) led to
improvement in the histological liver picture
together with significant restoration of hepatic
carbohydrate and protein contents. Chromium
reduced the elevated activities of AST and
ALT induced by CCl 4 , decreased the level of
total lipids and increased serum albumin.
These results proved that chromium had an
inhibitory effect against liver injury produced
by carbon tetrachloride.
Zoology Department, Faculty of Science, Menoufiya
University, Egypt.
Correspondance:
Saber A. Sakr
INTRODUCTION
Chromium is an essential trace mineral in
both human and animal nutrition since
enlarged body of evidence suggests that it
plays a crucial role in normal carbohydrate,
lipid and protein metabolism (Hepburn and
Vincent, 2003). Chromium works as a cofactor for increasing the action of insulin to
the optimal form, increases the uptake of
blood sugar in tissues and muscles cells and
increases the capacity of the cells to convert
blood sugar to glycogen (Frausto and
Williams, 1991; Lukaski, 1999). Mahmoud
(2002) indicated that chromium picolinate
significantly decreased the plasma levels of
total cholesterol (TC), triglyceride (TG), low
density lipoprotein cholesterol (LDL–C), very
low density lipoprotein cholesterol (VLDL–C),
and athreogenic index while significantly
increased the levels of high density
lipoprotein cholesterol (HDL- C).
Carbon tetrachloride toxicity has resulted
in many cases of poisoning by inhalation,
ingestion, or absorption (Sullivan and
Krieger, 1992). Prolonged exposure to carbon
tetrachloride
induced
histopathological
features such as inflammatory leucocytic
infiltration, necrosis, fibrosis, cirrhosis and
sometimes may lead to tumours. A single
dose of CCl 4 may lead to centrizonal necrosis
and steatosis (Recknagel and Goshal, 1966).
Pierce et al. (1987) found that poisoning by
CCl 4 induced toxic injury to both liver and
kidney.
Hepatic
damage
may
be
overshadowed
by
acute
renal
tubular
necrosis, leading to renal oliguria of many
species. Histochemical alterations such as
reduction of hepatic carbohydrates and
proteins were found to be induced by CCl 4
(Sakr et al., 2000). The present work aimed
to study the possible action of chromium
administration
against
hazardous effect
induced by CCl 4 .
MATERIAL AND METHODS
Healthy adult male albino mice Mus
musculus, approximately three months old
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92
weighing 20 ± 5 g were used in the present
study. All animals were kept in the laboratory
under constant conditions of temperature (24
± 3ºC) and humidity (57 ± 5 %) , at a dark light cycle 12 / 12 hrs. for at least one week
before and throughout the experimental work
.Animals were maintained on a standard
rodent diet and water was available ad
libitum. Animals were divided into 4 groups:
1- Control group:Animals of this group were divided into
two further subgroups: i- Subgroup (A): Animals of this subgroup
were given distilled water orally at dose level
of 20 µl /kg bw daily and served as control to
chromium-treated animals.
ii- Subgroup (B): Mice of this subgroup were
intraperitoneally injected with 10 ml/kg body
weight olive oil (the used solvent for carbon
tetrachloride) three times a week (day after
day) for 5 , 7, and 9 weeks. This group
served the CCl 4 control group.
2- Chromium picolinate-treated group:
Animals of this group were orally
administrated with chromium picolinate daily
at dose level of 20 µg /kg bw according to
human therapeutic dose for a total 9 weeks.
3- Carbon tetrachloride-treated group:Animals of this group were injected
intraperitoneally with CCl 4 at dose level of
0.01 ml / g bw three times a week (day after
day) for a total of 9 weeks .
4- Chromium picolinate-CCl4 treated group:
Animals of this group were treated with
chromium picolinate and carbon tetrachloride
by the same doses of groups 2 and 3.
Animals of all groups were sacrificed after
5, 7, and 9 weeks of treatment. The liver
was removed and fixed either in alcoholic
Bouin’s fluid for histological studies and
histochemical
demonstration
of
carbohydrates or in 10% neutral formalin for
demonstration of proteins. Fixed materials
were dehydrated in alcohol, cleared in
xylene, embedded in paraffin wax and
sections of 5 micrometres thickness were
cut using rotary microtome. Slides were
stained with haematoxylin and eosin for
histological examination (Lillie and Fulmer,
1976) or by Masson’s Tri-chrome method for
staining collagen fibres (Masson, 1929).
Concerning
histochemical
studies,
polysaccharides were demonstrated by
Periodic Acid Schiff reagent (PAS) technique
(Hotchkiss, 1948).
For visualization of the total proteins,
mercury bromophenol blue method of Mazia
et al. (1953) was applied.
For biochemical studies, blood samples
were collected from the hepatic portal vein
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Egypt. J. Exp. Biol. (Zool.), 3: 91 – 100 (2007)
of control and treated animals. Sera were
separated by centrifugation and stored at
20º C. In the Liver Institute, Menoufiya
University, alanine transaminase (ALT),
aspartate transaminase (AST) (Gella et al.,
1985), serum albumin (Doumas et al., 1971)
and serum total lipids(Tietz, 1983) were
determined using a fully automatic Hitachi
911 analyzer (Tokyo, Japan). A commercial
Randox Kits (Randox Laboratories LTd.
Ardomore, Crumlin, United Kingdom) were
used. The results were analyzed statistically
using Student's "t" test.
RESULTS
A- Histological results:
There were no observable differences
between animals that administrated olive oil
(the solvent of the CCl 4 ) or chromium and
control animals received distilled water (Figs.
1 and 2).
Fig. 1. Section in the liver of a control mouse showing
central vein (CV), hepatocytes (H), blood sinusoids
(S), and Kupffer cells (K) × 400
Fig. 2. Section in the liver of a mouse treated with
chromium showing portal vein (PV), bile ductile
(BD), and normal hepatic architecture × 400
Treating animals with CCl 4 induced many
time-dependent histological alterations in the
hepatic tissues (Table 1). In the specimens
S a k r et al . , Effect Of Chromium On Carbon Tetrachlorid-Induced Hepatotoxicity …
inspected after 5 weeks of treatment with
CCl 4 , the normal structural organization of
the hepatic lobules was lost and some of
hepatocytes showed cellular degeneration
and inflammatory leucocytic infiltration. The
sinusoidal
spaces
were
widened
and
contained activated Kupffer cells.
93
parenchyma forming cirrhotic nodules (Fig.
5).
Table 1. Histological changes induced in the liver of mice
during the experimental periods
Leucocytic
infiltration
Cytoplasmic
vaculation
Congestion of
blood vessels
Fatty infiltration
Necrosis
Fibrosis
CCl4 +
Cr.
group.
Number of
animals
CCl4
group
Treatment
periods(weeks)
Animals groups
Control
group
Cr.
group
-
20
-
-
-
-
-
-
-
20
5
7
9
5
7
9
7(3)
6(4)
5(5)
10
10
9(1)
+++
+++
+++
+
++
++
-
-
-
-
-
+++
+++
+
+
+++
+++
+++
+
+
+
+++
+++
+
+
++
-
+++
+
( ): Dead animals; + Mild, ++ Moderate, +++ Severe
The histopathological changes of the liver
were more pronounced after 7 weeks of
treatment with CCl 4 where the intrahepatic
vessels were congested with blood. Kupffer
cells displayed a noticeable activation
.Hypertrophied nuclei were also noted (Figs 3
and 4) and a considerable number of
hepatocytes displayed a marked cytoplasmic
vacuolation (Fig. 4). In addition, CCl 4 resulted
in micro and macro-vesicular steatosis (Fig.
4).
Fig. 4. Section in the liver of a mouse treated with
CCl 4 for 7 weeks showing micro- and macro-steatosis
(arrow) × 400
Fig. 5. Section in the liver of a mouse treated with
CCl 4 for 9 weeks showing bridging fibrosis (F) extends
to hepatic parenchyma forming cirrhotic nodules (CN)
× 400
Fig. 3. Section in the liver of a mouse treated with
showing
hypertrophied
nuclei
(arrow),
CCl 4
congested and enlarged portal vein (PV) × 400
After 9 weeks of CCl 4 treatment, the
hepatocytes suffered from obvious injury. The
inflammatory
leucocytic
infiltration
was
increased, in comparison to the 7-weeks
exposed animals’ liver, and a large area of
the liver showed necrosis and internal
hemorrhage. Fibrosis and bridging fibrosis
were observed in liver of 3 animals out of 5
that extends from portal tract to the hepatic
Fig. 6. Section in the liver of a mouse treated with
both CCl 4 and chromium for 5 weeks showing
cytoplasmic vacuolation, gaint nucleus (G) and few
leucocytic infiltration (arrow) × 400
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94
Examination of liver sections, prepared
from animals injected with both CCl 4 and
chromium for 5 weeks showed somewhat
healthy appearance as the liver tissue
displayed a normal architecture. Some of the
hepatocytes appeared with giant nuclei;
leucocytic infiltration was diminished but
blood sinusoids were dilated (Fig. 6).
Administration of both CCl 4 (three times a
week) and Cr daily for 7 and 9 weeks
produced an advanced degree of protection
against the histopathological effects induced
by CCl 4 as represented by complete absence
of fibrosis except in one case in the 9 weektreated animals. The individual hepatocytes
restored their morphological organization to a
certain extend, their cytoplasm exhibited
almost negligible vacuolization, and their
membranes became more distinct. The nuclei
also mostly restored their normal appearance
and the binucleated cells were also met
within this preparation (Fig. 7).
Egypt. J. Exp. Biol. (Zool.), 3: 91 – 100 (2007)
time-dependent
reduction
in
the
carbohydrates reaching its maximum after 9
weeks in a large number of hepatocytes
especially in the cirrhotic area (Fig. 11).
Specimens examined after 9 weeks of
treatment with carbon tetrachloride and
chromium revealed restoration of most
glycogen in comparison to the same period of
carbon tetrachloride group (Fig. 12).
Fig. 8. Section in the liver of a control mouse showing
small amounts of wavy bundles of collagen around
the portal vein (PV) × 400
Fig. 7. Section in the liver of a mouse treated with
both CCl 4 and chromium for 9 weeks showing
binucleated cells (arrow) and improvement of the
hepatic tissue × 400
Concerning collagen fibres; in control and
chromium-treated animals collagen occurred
as wavy fibrils either singly or clustered
together in dense bundles around blood
vessels and sinusoids as shown in figure 8.
Administration of carbon tetrachloride three
times a week for 9 weeks resulted in high
proliferation of collagen fibres that appeared
in the form of thick bundles distributed in
most portions of the tissues denoting liver
cirrhosis with advanced fibrous septa of
scare tissue (Fig. 9).
B-Histochemical results:
The carbohydrate content in control
animals and those treated with chromium
applied as deeply-stained reddish granules
only in the cytoplasm as shown by PAS
reaction. These granules were confirmed to
be glycogen by Best's carmine method with
and without previous treatment with diastase
(Fig. 10). Treatment with CCl 4 induced a
http://www.egyptseb.org
Fig. 9 Section in the liver of a mouse treated with CCl 4
for 9 weeks exhibiting fibrosis and the fibrous septa
are broad and surround the hepatic blood vessels
× 400
There is no observable difference
between protein content of the control and
that of mice treated with chromium (Fig. 13).
Examination of liver sections of animals after
5
weeks
of
treatment
with
carbon
tetrachloride showed that most hepatocytes
appeared with a reduction of their proteinic
content. Specimens inspected after nine
weeks of treatment with CCl 4 revealed that
the cytoplasm of a large number of
hepatocytes was nearly devoid of protein
(Fig. 14). After nine weeks of treatment with
both chromium and CCl 4 , animals showed an
S a k r et al . , Effect Of Chromium On Carbon Tetrachlorid-Induced Hepatotoxicity …
95
elevation in the proteinic content, in
comparison with the same period of carbon
tetrachloride- treated group (Fig. 15).
Fig. 10. Section in the liver of control mouse showing
normal distribution of glycogen in the cytoplasm of
hepatocytes. The nuclei exhibit negative stain
Fig. 13. Section in the liver of control mouse showing
normal dense proteinic content with normal
distribution in all hepatocytes × 400
× 400
Fig. 11. Section in the liver of a mouse treated with
CCl 4 for 9 weeks showing cirrhotic area contains
low amounts of glycogen in the cytoplasm of most
hepatocytes and fibrosis (F) × 400
Fig. 12. Section in the liver of a mouse treated with
both chromium and CCl 4 for 9 weeks showing
limited decrease in glycogen content × 400
Fig. 14. Section in the liver of a mouse treated with
CCl 4 for 9 weeks showing a large number of
hepatocytes that nearly devoid of protein
× 400
Fig. 15. Section in the liver of a mouse treated with
chromium and CCl 4 for 9 weeks showing an increase
in proteinic materials in the hepatocytes compared
with those of the CCl 4 -treated mice × 400
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Egypt. J. Exp. Biol. (Zool.), 3: 91 – 100 (2007)
96
Me a n ±S D
50.6 ± 7.2
38.8±3.2
7 W eeks
41.2± 3.3
40 ± 3.6
55.4±7.3
41.8±3.3
9 W eeks
40.6± 1.5
39.8 ± 3.7
67.8± 8.7 ª
51.6±7.4
5 W eeks
42.4± 6.2
35.2± 5.72
69.4 ± 8.65
45.8±7.4
7 W eeks
41.8± 7.2
39.4 ± 6.1
1 0 7 . 8 ± 1 2 . 3ª
51.2±7.8
9 W eeks
39.2± 5.9
33.0 ± 5.5
1 4 2 . 4 ± 1 8 . 2ª
61.2±8.2
n = 5 animals for each group.
a: Significant increase (p < 0.05) in comparison with
control group.
3 - Changes in serum albumin level:Results in table 4 shows that there is a
significant decrease in albumin in animals
treated with carbon tetrachloride more than
those of the control and chromium-treated
groups. On the other hand, the albumin level
in animals given both chromium and carbon
tetrachloride exhibited a marked elevation
compared to treated groups.
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3.9 ± 0.13
7 W eeks
4.7 ± 0.1
4.5 ± 0.3
1.8 ± 0.0 ª
3.8 ± 0.2
9 W eeks
4.6 ± 0.2
4.1 ± 0.4
1.4 ± 0.1 ª
3.8 ± 0.2
n = 5 animals for each group.
a: Significant decrease (p < 0.05) in comparison with
control group.
4- Changes in serum total lipids level:
Data in table 5 shows that treating
animals with carbon tetrachloride induced
significant increase in serum total lipids after
5, 7, and 9 weeks of treatment compared with
animals of control and chromium-treated
groups. Animals treated with both chromium
and carbon tetrachloride showed reduction in
their sera’s total lipids in comparison of those
given only CCl 4 in all treatment periods.
Table 5: Effect of chromium (Cr) and carbon
tetrachloride (CCl 4 ) on serum total lipids level (mg /
dl)
Cr + CCl 4
g ro u p
Me a n ±S D
Me a n ± S D
2.1 ± 0.2
CCl 4
g ro u p
Me a n ± S D
Cr +
CCl4
g ro u p
Cr
g ro u p
Me a n ± S D
CCl4
g ro u p
Co n t ro
l g ro u p
T re a t me n t
P e rio d
Me a n ± S D
Me a n ± S D
4.4 ± 0.1
Cr
g ro u p
Table 3. Effect of chromium (Cr) and carbon
tetrachloride
(CCl4)
on
serum
alanine
aminotransferase (ALT, U/l)
Me a n ± S D
4.5 ± 0.25
Co n t ro l
g ro u p
2Changes
in
serum
alanine
aminotransferase (ALT) activity:Table 3 shows the effective action of
chromium in decreasing the ALT level in sera
of mice given carbon tetrachloride.
Both
control and chromium-treated group showed
no significant differences in serum ALT
activity.
Me a n ± S D
5 W eeks
T re a t me n t
P e rio d
n = 5 animals for each group.
a: Significant increase (p < 0.05) in comparison
with control group.
Cr + CCl4
g ro u p
Me a n ± S D
39 ± 3.9
CCl4
g ro u p
Me a n ± S D
39.2± 3.2
Cr
g ro u p
Cr +CCl 4
g ro u p
CCl 4
g ro u p
Cr
g ro u p
Co n t ro l
g ro u p
T re a t me n t
P e rio d
Me a n ± S D
5 W eeks
Co n t ro l
g ro u p
Table 2: Effect of chromium (Cr) and carbon
on
serum
aspartate
tetrachloride
(CCl 4 )
aminotransferase (AST, U/l)
Table 4. Effect of chromium (Cr) and carbon
tetrachloride (CCl 4 ) on serum albumin level (g/dl)
T re a t me n t
P e rio d
C-Biochemical analysis:
1Changes
in
serum
aspartate
aminotransferase (AST) activity:
Data in table 2 shows that there was no
significant difference in serum AST activity
between control mice and those treated with
chromium in all treatment periods. Sera of
animals treated with CCl 4 showed a
significant increase in serum AST activity
especially after 9 weeks of treatment
.Animals treated with both chromium and
carbon tetrachloride had a noticeable
decrease in AST activity compared with
animals received CCl 4 alone.
Me a n ± D
Me a n ± S D
Me a n ± S D
Me a n ± S D
5 W eeks
348±10.3
330 ± 8.9
392.4± 13.5
3 4 2 . 4 ± 1 0.3
7 W eeks
345± 9.9
330 ± 8.9
494.6± 16.8
418± 13.0
9 W eeks
355±11.2
356 ± 11.3
864.6±25.6ª
4 5 4 . 8 ± 1 4.7
n = 5 animals for each group.
a: Significant increase (p < 0.05) in comparison with
control group.
DISCUSSION:
Results obtained in the present work
revealed that treating male mice with carbon
tetrachloride
induced
histological,
histochemical and biochemical alterations in
the liver. Also, the present investigation
showed the possible protective effect of
chromium on the liver injury induced by
carbon tetrachloride.
In the present work early hepatic damage
was confirmed in mice treated with CCl 4 ; this
damage appeared in the form of an
inflammatory leucocytic infiltration, widening
of the sinusoids, activation of Kupffer cells,
loss of normal hepatic tissue architecture and
disappearance
of
normal
organization.
Similar results were obtained by Edwards et
al. (1993) who found an inflammatory
response occurred when Kupffer cells were
activated by the free radicals and secretes
cytokines that attract and activate neutrophils
thereby enhancing the liver injury. Plaa and
S a k r et al . , Effect Of Chromium On Carbon Tetrachlorid-Induced Hepatotoxicity …
Hewitt (1982) observed that liver injury
including marked alteration of the entire liver
structures with degenerative and proliferative
changes were observed in the different forms
of CCl 4 -induced cirrhosis.
In the present study a remarkable
cytoplasmic vacuolation of the hepatocytes
was observed in mice treated with CCl 4 . This
result was in agreement with Sakr et al.
(2000) who found cytoplasmic vacuolation of
the
hepatocytes
after
treating
rats
subcutaneously with CCl 4 at dose level of 0.1
ml/100 g body weight for 10 days. The
authors examined lipids content in the liver of
rats after treatment with CCl 4 and recorded
that rats subcutaneously injected with CCl 4 at
dose level of 0.1/ 100 g rat for 10, 50, 90,
and 130 days, exhibited accumulation of fat
droplets which became comparatively larger
in size and increased in number with the
increase of liver injury. Moreover, Brody et al.
(1961) attributed the fatty changes in the liver
to the excessive mobilization of free fatty
acids from the fat depots induced by the
lipolytic effects of the increased circulating
catecholamines
and
the
centrilobular
necrosis to the catecholamines-induced
decrease in hepatic blood flow. Plaa and
Hewitt (1982) reported that liver injury also
causes
the
accumulation of abnormal
amounts of fat in the parenchymal cells.
After 9 weeks of treating mice with CCl 4 ,
the histopathological alterations appeared to
be increased, followed by increase of
interstitial collagen in extracellular matrix.
These results were in agreement with those
indicated by Pierce et al. (1987) and Louis et
al. (1998) after treating mice with CCl 4
subcutaneously for 45 days and found a
reversible acute centrilobular liver necrosis,
followed by fibrosis.
Histochemical results obtained in the
present work showed that glycogen content
was depleted markedly in the hepatocytes
after administration of CCl 4 ; such depletion
was increased with progression of time
reaching its maximum after 9 weeks of
treatment. Such decrease either could be
attributed to the increasing stress on
hepatocytes or to loss of liver cells ability to
store glycogen as a result of CCl 4 toxicity.
These results agree with those of Sakr et al.
(2000) who found a slight decrease in
glycogen after 10 days of treatment with CCl 4
but such depletion was increased after 50
days, then glycogen depletion become
prominently remarkable after 90 days; an
almost complete absence of glycogen was
observed at 130 days of treatment.
In this work, a time-dependent reduction
in total protein content was recorded in the
liver cells of mice after CCl 4 toxicity. This
97
reduction come in accordance with results
obtained by Sakr et al. (2000) who observed
a decrease in the protein content in rats'
liver after injection with carbon tetrachloride.
Such decrease became more apparent after
130 days of treatment.
Also, Rhoden et al. (2000) reported
significant decrease (of 18%) in total hepatic
protein and in RNA content (37%) in rats
given CCl 4 . Impairment of protein synthesis in
CCl 4 hepatotoxicity was represented by Lamb
et al. (1984). They stated that CCl 4 caused
membrane lipids peroxidation, altered lipids
metabolism and decrease protein synthesis in
the
injured
hepatocytes.
In
addition,
intoxication with CCl 4 increases the ratio of
the liver weight to body weight and decreases
the concentration of microsomal protein
which was dependent upon the dose of CCl 4 .
The increasing percentage in the liver weight
was equal to the decreasing percentage in
the concentration of microsomal protein.
Boger et al. (1987) concluded that the drug
metabolism observed in CCl 4 poisoning
resulted primarily from an inactivation of the
microsomal enzymes rather than from a loss
of enzymatic protein
Biochemical determination of aspartate
amino-transferase (AST) and alanine aminotransferase (ALT) levels in sera of animals
treated with CCl 4 in the present work
indicated significant increase (p < 0.05).
Similar results were obtained by some
researches. Wang et al. (1996) observed that
a single intraperitoneal injection of CCl 4 (20
% in olive oil/g bw) caused an increase in
serum aspartate amino- transferase and
alanine amino- transferase levels.
Pablo and Yesenia (2003) discovered that
liver injury induced by CCl 4 in Wistar rats was
accompanied by an increase in serum
enzyme activities of alkaline phosphatase
(ALP),
gamma-glutamyl
transepeptidase
(gamma-GTP), aspartate aminotransferase
(AST), alanine aminotransferase (ALT) and
bilirubin concentrations.
Tirkey et al. (2005) observed that when
CCl 4 (2 ml/kg) administrated subcutaneously
for one week to male rats a marked liver
damage which represented by markedly
elevated levels of serum ALT and AST
activities were observed.
Ryeom et al. (2004) injected adult New
Zealand white rabbits with CCl 4 three times a
week for three weeks. The results showed
that liver enzymes (ALT and AST) levels were
significantly
increased,
as
AST
was
increased from 39.8 ± 5.2 IU/l to 138.4 ± 50.5
IU/l while ALT was increased from 59.1±11.7
IU/l to 172.0 ± 71.6 IU/l .
In the present work, serum albumin was
significantly decreased
in CCl 4 -intoxicated
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98
mice, a result which matched with Okuyama
et al. (1975) who injected adult male rats
with CCl 4 and reported that in normal liver,
albumin synthesis averaged 5.6 mg/hr/100g
body weight, while in CCl 4 - intoxicated liver,
albumin
synthesis
decreased
to
4.6
mg/hr/100 g bw. From these results, albumin
synthesis rate decreases in accordance with
the progress of the liver injury.
Torres – Duran et al. (1999) assessed
liver and serum lipid levels changes in rats
treated with a single intraperitoneal dose of
CCl 4 for 4 days and indicated that VLDL,
LDL, HDL, triglyceride, and cholesterol
displayed marked increases.
In the present work, Chromium was
tested for its protection against CCl 4
hepatotoxicity. Administration of chromium to
normal healthy mice did not cause any
histological or histochemical changes in the
liver. These results were in accordance with
the findings of Guan et al. (2000) and Rai et
al. (2005) who decided the safety of
chromium administration. Shinde and Goyal
(2003) found that chronic treatment with
chromium picolinate, did not alter the normal
function or morphology of control rats.
In the present work, chromium exerted
reducing effects on the histopathological
changes induced by CCl 4 injection as there
were a marked decrease of inflammatory
leucocytic infiltration, steatosis, necrosis and
fibrosis. Moreover, it reduced cases of
cirrhosis that has been recorded by treatment
with CCl 4 alone. Histochemically, chromium
succeeded to return both carbohydrates and
proteins to nearly normal quantities after
being decreased from CCl 4 injection.
The effect of administration of trivalent
chromium [Cr(III)] to mice and the activation
of carbon tetrachloride (CCl 4 ) to form
trichloromethyl radicals (CCl3) in the liver
were studied by Tezuka et al. (1991). The
lipid peroxidation in liver microsomes induced
in vitro by CCl 4 in the presence of NADPH
was decreased by the preadminstration of Cr
(III) to mice. The activity of NADPHcytochrome C reductase, which presumably
catalyzes the formation of CCl3 from CCl 4 in
liver microsomes was depressed by Cr (III)
administration and kept at a level lower than
that of the control group for at least 2 hr after
CCl 4 dosing.
In vitro study using primary cultured
mouse hepatocytes was performed to discuss
the effect of Cr (III) and several other trace
metals, Cr (VI), Mn (II), Zn (II), Co (II), Cu
(II), Ni (II) and Ga (III) on acute liver damage
induced by CCl 4 . The LDH activity 60 min
exposure
increased
doseafter
CCl 4
dependently with CCl 4 concentrations in all
the trace metal pretreatment groups, except
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Egypt. J. Exp. Biol. (Zool.), 3: 91 – 100 (2007)
for the Cr (VI) pretreatment group, which
showed a significant protective effect even
after 30 min of CCl 4 exposure. LDH leakage
was not observed 10 min after CCl 4 exposure
at 3 or 5 mM, while lipid peroxidation was
increased
dose-dependently
with
CCl 4
concentrations in all groups except the Cr
(VI) pretreatment group, in which the
production
of
peroxidated
lipid
was
significantly inhibited. The results suggested
that the in vitro protective effect of
pretreatment with Cr (VI) was due to a rapid
reduction of Cr (VI) to Cr (III); it therefore
suggests that Cr (III) contributes to protective
effect on CCl 4 -induced hepatotoxicity (Tezuka
et al., 1995).
Onderci et al. (2005) examined the
effects of chromium picolinate in Japanese
quail (Coturnix coturnix japonica) subjected
to high environmental temperature and the
obtained data showed that supplementation
with chromium picolinate ameliorated the
deleterious effect of stress. Performance,
carcass characteristics, levels of oxidative
stress markers, serum cholesterol and
glucose concentrations were improved after
treatment with chromium picolinate.
Bernao et al. (2004) evaluated the effects
of three different levels of dietary chromium
(100, 200 and 500 µg/day) in the form of
chromium picolinate on growth and protein
use in weaned rats. No significant effect of
chromium picolinate on body weight gain,
food intake, or food conversion rate was
observed. Elevated doses of chromium
picolinate increased muscle mass, either by
stimulating protein anabolism by activation of
insulin by chromium or by lowering protein
degradation.
The mechanism of chromium's possible
glucose-regulatory
activity
is
not
well
understood, but there are some theories
attempting to explain such phenomenon. It is
thought that the possible action of chromium
on
the
control
of
blood
glucose
concentrations is the potentiation of insulin.
One proposed mechanism involves increased
insulin binding, increased insulin receptor
number and increased insulin receptor
phosphorylation of the insulin receptor, which
is
associated
with
increased
insulin
sensitivity (Donaldson and Barreras, 1966).
In summary, results obtained in this work
demonstrated that
chromium ameliorated
the hepatotoxicity of CCl 4 in mice .This may
be attributed to decrease of lipid peroxidation
in liver microsomes induced by CCl 4 (Tezuka
et al., 1991) or/ and it acts as an antioxidant
that scavenges free radicals and that it could
restore the normal liver function (Hanna et
al., 1993).
S a k r et al . , Effect Of Chromium On Carbon Tetrachlorid-Induced Hepatotoxicity …
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: ‫ﺗﺄﺛﻴﺮ اﻟﻜﺮوم ﻋﻠﻰ راﺑﻊ آﻠﻮرﻳﺪ اﻟﻜﺮﺑﻮن اﻟﻤﺤﺪث ﻟﻠﺴﻤﻴﺔ اﻟﻜﺒﺪﻳﺔ ﻓﻲ اﻟﻔﺌﺮان‬
‫دراﺳﺎت ﻧﺴﻴﺠﻴﺔ وﻧﺴﻴﺠﻮآﻴﻤﻴﺎﺋﻴﺔ‬
‫ راﻧﻴﺎ ﻋﺒﺪ اﻟﻜﺮﻳﻢ ﺷﻌﻴﺮ‬،‫ هﺎﻧﻰ ﻋﺒﺪ اﻟﺤﻤﻴﺪ ﻋﺒﺪ اﻟﺴﻤﻴﻊ‬،‫ﺻﺎﺑﺮ ﻋﺒﺪ اﻟﺮﺣﻤﻦ ﺻﻘﺮ‬
‫ ﺟﺎﻣﻌﺔ اﻟﻤﻨﻮﻓﻴﺔ‬،‫ آﻠﻴﺔ اﻟﻌﻠﻮم‬،‫ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﻴﻮان‬
‫ وﻟﻢ ﺗﺆدي اﻟﻤﻌﺎﻣﻠﺔ ﺑﻤﺎدة اﻟﻜﺮوم إﻟﻰ ﺗﻐﻴﺮ ﻳﺬآﺮ ﻓﻲ ﺗﺮآﻴـﺐ‬،‫هﺬا‬
‫اﻟﻜﺒﺪ اﻟﻨﺴﻴﺠﻲ أو اﻟﻨﺴﻴﺠﻮآﻴﻤﻴﺎﺋﻲ أو ﺣﺘـﻰ ﻓـﻲ آﻴﻤﻴـﺎء اﻟﻜﺒـﺪ‬
‫ راﺑــﻊ آﻠﻮرﻳــﺪ‬:‫ ﺑــﻞ أن ﻣﻌﺎﻣﻠــﺔ اﻟﺤﻴﻮاﻧــﺎت ﺑﻜــﻼ اﻟﻤــﺎدﺗﻴﻦ‬،‫اﻟﺤﻴﻮﻳــﺔ‬
‫اﻟﻜﺮﺑــﻮن واﻟﻜــﺮوم ﻗــﺪ ﺣــﺴﻨﺖ ﻣــﻦ اﻟــﺼﻮرة اﻟﻨــﺴﻴﺠﻴﺔ ﻟﻠﻜﺒــﺪ‬
‫و ﺗــﺴﺒﺒﺖ ﻓــﻲ اﺳــﺘﻌﺎدة اﻟﺤﺎﻟــﺔ اﻟﻄﺒﻴﻌﻴــﺔ ﻟﻜــﻞ ﻣــﻦ اﻟﻤــﻮاد‬
‫ آﻤــﺎ ﻗﻠﻠــﺖ ﻣــﻦ اﻷﻧــﺸﻄﺔ‬،‫اﻟﻜﺮﺑﻮهﻴﺪراﺗﻴــﺔ و اﻟﺒﺮوﺗﻴﻨــﺎت اﻟﻜﻠﻴــﺔ‬
‫ اﻟﻤﺤﺪﺛﺔ ﺑﻤﺎدة راﺑﻊ آﻠﻮرﻳﺪ اﻟﻜﺮﺑﻮن‬AST ‫ و‬ALT ‫اﻟﻤﺘﺰاﻳﺪة ﻹﻧﺰﻳﻤﻲ‬
‫ ﻣﻤﺎ ﻳﺪل ﻋﻠـﻰ‬،‫و ﻗﻠﻠﺖ ﻣﻦ اﻟﺪهﻮن اﻟﻜﻠﻴﺔ و رﻓﻌﺖ ﻣﻦ اﻷﻟﺒﻴﻮﻣﻴﻦ‬
‫أن ﻟﻠﻜﺮوم ﺗﺄﺛﻴﺮ واﻗﻲ ﺿـﺪ اﻟـﺴﻤﻴﺔ اﻟﻜﺒﺪﻳـﺔ اﻟﻤﺤﺪﺛـﺔ ﺑﻤـﺎدة راﺑـﻊ‬
.‫آﻠﻮرﻳﺪ اﻟﻜﺮﺑﻮن‬
:‫اﻟﻤﺤﻜﻤﻮن‬
‫ ﻋﻠﻮم ﻋﻴﻦ ﺷﻤﺲ‬،‫ ﻣﻨﻴﺮ اﻟﺠﻨﺰوري ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﻴﻮان‬.‫د‬.‫أ‬
‫ ﻋﻠﻮم ﻃﻨﻄﺎ‬،‫ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﻴﻮان‬
‫ ﻓﺆاد ﻋﻔﻴﻔﻲ‬.‫د‬.‫أ‬
http://www.egyptseb.org
‫ﻳﺴﺘﺨﺪم ﻣﺮآﺐ راﺑﻊ آﻠﻮرﻳﺪ اﻟﻜﺮﺑﻮن ﺑﻜﺜﺮة ﻓـﻲ‬
‫ وﻗﺪ وﺟﺪ أن ﺣﻘﻦ هﺬﻩ ﻟﻤـﺎدة ﻓـﻲ‬.‫اﻟﻌﺪﻳﺪ ﻣﻦ اﻟﺼﻨﺎﻋﺎت‬
/ ‫ ﻣﻠﻠﻴﻠﺘـﺮ‬0.01 ‫اﻟﺘﺠﻮﻳﻒ اﻟﺒﺮﻳﺘﻮﻧﻲ ﻟﻠﻔﺌﺮان ﺑﺠﺮﻋﺔ ﺗﻌﺎدل‬
9 ، 7 ، 5 ‫ﺟﺮام ﻣﻦ وزن اﻟﺠﺴﻢ ﺛﻼث ﻣﺮات أﺳﺒﻮﻋﻴﺎ ﻟﻤﺪة‬
‫أﺳﺎﺑﻴﻊ ﻗـﺪ أﺣـﺪﺛﺖ ﺗﻐﻴـﺮات ﻧـﺴﻴﺠﻴﺔ و ﻧـﺴﻴﺠﻮآﻴﻤﻴﺎﺋﻴﺔ‬
‫ و ﻇﻬﺮت اﻟﺘﻐﻴﺮات اﻟﻨﺴﻴﺠﻴﺔ ﻓـﻲ‬.‫هﺎﺋﻠﺔ ﻓﻲ ﺧﻼﻳﺎ اﻟﻜﺒﺪ‬
، ‫ ﺗﺨﻠﻞ ﻟ ﻠﺨﻼﻳـﺎ اﻻﻟﺘﻬﺎﺑﻴـﺔ‬، ‫ﺻﻮرة اﺣﺘﻘﺎن ﻟ ﻸوﻋﻴﺔ اﻟﺪﻣﻮﻳﺔ‬
‫وﻓﺠﻮات ﻓﻲ اﻟﺴﻴﺘﻮﺑﻼزم و ﻇﻬﻮر اﻟﻘﻄﻴﺮات اﻟﺪهﻨﻴـﺔ ﻣـ ﻊ‬
‫ و ﻣـــــﻦ اﻟﻨﺎﺣﻴـــــﺔ‬. ‫ﺗﻠﻴـــــﻒ ﻟ ﻠﻜﺒـــــﺪ وأﺧﻴـــــﺮا ﺗـــــﺸﻤﻌﻪ‬
‫ ﺗﺴﺒﺐ ﺣﻘﻦ راﺑﻊ آﻠﻮرﻳﺪ اﻟﻜﺮﺑﻮن ﻓـﻲ‬، ‫اﻟﻨﺴﻴﺠﻮآﻴﻤﻴﺎﺋﻴﺔ‬
‫ﻧﻘﺺ اﻟﻤﻮاد اﻟﻜﺮﺑﻮهﻴﺪراﺗﻴﺔ واﻟﺒﺮوﺗﻴﻨﺎت اﻟﻜﻠﻴﺔ ﻓﻲ ﺧﻼﻳـﺎ‬
‫ و اﻟﺪهﻮن‬AST ‫ و‬ALT ‫اﻟﻜﺒﺪ ﻓﻲ ﺣﻴﻦ زاد ﻧﺸﺎط إﻧﺰﻳﻤﻲ‬
،‫اﻟﻜﻠﻴﺔ ﻓﻲ اﻟﻮﻗﺖ اﻟﺬي ﺗﺴﺒﺐ ﻓﻴﻪ ﻓﻲ ﻧﻘﺺ اﻷﻟﺒﻴﻮﻣﻴﻦ‬
. ‫و آﺎﻧﺖ آﻞ هﺬﻩ اﻟ ﺘﻐﻴﺮات ﻣﺮﺗﺒﻄﺔ ﺑﺎﻟﻮﻗﺖ‬