Evaluation Of
Protective Role Of Thiola Against Carbon Tetrachloride-Induced Histochemical
Changes In The Rat Liver
SABER
A. SAKR1, ESAM I. AGAMY1, MARSEL N. BOULOS2 AND ENAYAT A. OMARA2
1- Department
of Zoology, College of Science,
Menoufia University, Egypt
2- Department
of Pathology, National Research Center,
Cairo,
Egypt
تأثير
الثيولا على التغيرات الهستوكيميائية المستحثة بواسطة رابع كلوريد الكربون في كبد
الفئران
تضمنت هذه الدراسة تأثير مركب الثيولا وهو أحد مركبات السلفهيدريل على التغيرات الهستوكيميائية التي يحدثها رابع كلوريد الكربون في كبد الفئران. أظهرت النتائج أن معاملة الحيوانات برابع كلوريد الكربون لمدة 50 ، 90 ، 130 يوما أدت إلى انخفاض كميات الجليكوجين، المواد البروتينية انخفاضا ملحوظاً في الخلايا الكبدية، بينما زادت كمية الدهون ونشط أنزيم الفوسفاتيز القاعدي. أدى حقن الفئران بمركب الثيولا بعد المعاملة برابع كلوريد الكربون إلى تحسن ملحوظ في المحتوى الهستوكيميائي للكبد فقد ظهر الجليكوجين والبروتينات الكلية بصورة شبه طبيعية بعد إنتهاء الحقن بالثيولا، ونقصت كمية الدهون وأنخفض نشاط أنزيم الفوسفاتيز القاعدي. وقد أستدل من هذه الدراسة أن للثيولا تأثيرا واقيا ضد التغيرات المرضية التي يحدثها رابع كلوريد الكربون.
The effect of the sulfhydryl compound (thiola) on the
histochemical changes induced by carbon tetrachloride (CCl4) in the liver of
rat was investigated after different periods of treatment. A marked decrease of
glycogen and total proteins was noticed in the hepatic cells of CCl4 treated
rats after 90 and 130 days of treatment. On the other hand, fatty infiltration
was markedly observed in the treated liver and increased by increasing the
duration of treatment. A marked increase in the activity of alkaline
phosphatase appeared in the liver of treated animals, especially in the
degenerated areas. Animals treated with CCl4 followed by injection of thiola
for 50, 90 and 130 days showed a significant improvement in the liver
histochemical parameters. The degree of improvement depended on the period of
thiola treatment.
Keywords: Liver, carbon tetrachloride, thiola, histochemistry.
INTRODUCTION
Hepatic injury
induced by chemicals has been recognized as one of the most toxicological
problems. Carbon tetrachloride (CCl4) is one of the hydrocarbons that has a
wide spread use in various industries as solvent (El-Dossouky et al. 1978). It
is also used in medicine as vermifuge in the treatment of hookworm disease. On
the other hand, exposure to CCl4 by inhalation, ingestion or absorption through
the skin resulted in many cases of poisoning (Boger et al. 1987). CCl4 is a potent hepatotoxin in a
variety of experimental animals and man. It induced necrosis and cirrhosis
(Ishil et al. 1984). Prolonged administration of CCl4 leads to fibrosis,
cirrhosis and hepatic carcinoma (Zimmerman & Ishak, 1978).
Many
sulfhydryl compounds have been used extensively for protection against
injurious effect of many toxic substances as well as ionizing radiations. Among
these compounds found in the living body are cysteine, glutathione and
ergothionine. Thiola
(2-mercaptoproionylglycine) is one of the sulfhydryl compounds that contains a
weakly-bonded sulfhydryl radical, SH. It has been used for protection against
ionizing radiation without any serious toxicity (Sughora et al. 1970). Thiola
has been also proven to be effective in management of various forms of liver
dysfunction (Tsuji & Nagashima 1981; Ichida et al. 1982; Sakr 1993). It was
effective in modulating hepatic alterations following repeated administration
of fibrotic agent as CCl4 and ethanol (Torriell et al. 1981). In continuation to
our previous study on the effect of thiola on histological changes induced in
the rat liver by CCl4 (Sakr et al. 1994), the present work was planned to
evaluate the possible preventive role of thiola on histochemical alterations
produced by CCl4 in the liver of male albino rats.
MATERIALS AND METHODS
Adult male albino
rats (Rattus norvigus) weighing 70-80 gms were used in the present study. They
were fed a standard rodent chow (pellets, Egyptian Ministry of Agriculture) and
water was given ad libitum. Animals (110 rats) were divided into four groups.
Group I Animals of
this group (25 rats) served as control.
Group II Animals of this
group (30 rats) were injected subcutaneously with 0.1ml CCl4
(32 gm/L) twice weekly for 130 days.
Group III Animals of this group (30
rats) received the same dose level of CCl4 of those in-group II followed by
daily intraperitoneal injection of thiola (1 mg/100gm body weight) for 130
days. Thiola was dissolved in saline and neutralized with NaHCO3 before use.
Group IV Animals of this group (25
rats) were injected with thiola only.
Eight
animals were selected randomly from each group after 50, 90 and 130 days of
treatment, sacrificed and prepared for histochemical examination. Small pieces
of the liver were quickly removed and fixed in Carnoy’s fluid. Fixed materials
were embedded in paraffin wax and sections of 5 microns were cut.
Polysaccharide materials were demonstrated using periodic acid - Shiff’s
technique PAS (Hockiss 1948). Total proteins were demonstrated by mercury
bromophenol blue method (Mazia et al. 1953). Frozen sections stained by oil
blue N method were used for demonstration of lipids (McManus & Mowry,
1960). Gomori’s method was used for demonstration of alkaline phosphatase
(Moussa et al. 1984).
RESULTS
GLYCOGEN
In
control rats the total carbohydrates exist in the form of deeply stained
reddish granules in the cytoplasm of the hepatic cells, as shown by PAS method.
All these positively stained materials have been proved to be glycogen as
verified by Best’s carmine staining with and without previous treatment with
diastase. The nuclei gave a negative reaction (Fig. 1).
Liver
of animals treated with CCl4 for 50 days showed noticeable decrease in glycogen
in the cytoplasm of most hepatocytes and the hepatic lobules lost their normal
configuration (Fig. 2). Such reduction of glycogen markedly appeared in
the liver of animals treated for 90 and 130 days (Fig. 3). Rats treated
with CCl4 followed by injection of thiola revealed marked degree of improvement
in glycogen contents of the hepatocytes and the liver tissue restored most of
its normal structure. Such restoration was seen after 130 days of
treatment (Fig. 4).
LIPIDS
Using
oil blue method, lipid materials were stained blue. In the control rat, the
liver showed very little lipid inclusions in the form of few blue droplets as
well as very fine granules scattered randomly in the cytoplasm (Fig. 5).
Animals treated with CCl4 showed an increase in lipid droplets. After 50
days of treatment fat droplets were markedly increased in the cytoplasm of the
affected hepatocytes. The distribution of fat droplets in some rats was in the
mid peripheral zone of liver lobules and in others the droplets were
distributed randomly in all the lobules (Fig. 6).
Fig. 1. Section in liver of a control rat showing PAS -
positive inclusions (glycogen) in the hepatocytes. x 300. Fig. 2.
Section in liver of a rat treated with CCl4 for 50 days showing a decrease of
glycogen. x 300. Fig. 3. Section in liver of a rat treated with CCl4 for
90 days showing noticeable decrease of glycogen. x 300. Fig. 4. Section
in liver of a rat treated with CCl4 followed by thiola for 130 days showing
restoration of glycogen contents in most cells. x 300.
In the hepatocytes
of animals treated with CCl4 for 90 and 130 days, the extent of visible fat
accumulation varied from minute droplets scattered in the cytoplasm of few
cells to distention of the entire cytoplasm of the most cells by coalesced
droplets (Fig. 7). Animals treated with CCl4 followed by thiola showed
gradual decrease in lipid inclusion in the hepatocytes. After 130 days of
injection with thiola, these inclusions were markedly decreased as compared
with those treated with CCl4 alone (Fig. 8).
TOTAL PROTEINS
The
proteinic materials in the liver cells were demonstrated by bromophenol blue
method. In the control rats, they appeared in the form of small bluish
irregular particles which were sometimes closely packed together making blue
irregular dense bodies against a weakly to moderately stained ground cytoplasm
(Fig. 9).
Examination
of rat liver after treatment with CCl4 for 50 days showed moderate decrease in
its proteinic contents (Fig. 10). After administration of CCl4 for 90
and 130 days, the hepatocytes designated a high decrease in their protein
materials (Fig. 11). A slight decrease in protein content was observed
in the majority of hepatic cells of animals treated with CCl4 followed by
thiola
(Fig. 12).
ALKALINE PHOSPHATASE
Gomori’s
method was used to demonstrate alkaline phosphatase histochemically in tissue
fixed in cold acetone. In the liver of control rats, the nuclei of the
peripheral cells of the lobule showed a weak alkaline phosphatase activity
while the central part showed negligible response. The enzymatic activity in
the wall of blood vessels and sinusoids was mild (Fig. 13).
After
administration of CCl4 for 50 days the hepatic cells showed an increase of
alkaline phosphatase compared with the controls (Fig. 14). A marked increase
in the activity of alkaline phosphatase was observed in the liver of rats
treated for 90 and 130 days. Marked reaction was seen in the degenerated areas
(Fig. 15). An improvement in the concentration and quantity of alkaline
phosphatase content was observed in the liver of animals treated with CCl4 and
thiola, and such partial improvement was apparent after 90 days (Fig. 16).
Fig. 5. Section in liver of a control rat stained by oil
blue N stain showing distribution of lipid granules . x 300. Fig. 6.
Section in liver of a rat treated with CCl4 for 50 days showing lipid droplets
distributed in most of the hepatocytes. x 300. Fig. 7. Section in liver
of an animal treated with CCl4 for 130 days showing heavy fat accumulation . x
300. Fig. 8. Section in liver of a rat treated with CCl4 and thiola for
130 days showing remarkable depletion in lipid droplets. x 300.
Fig. 9. Section in liver of a control rat stained by
bromophenol blue showing strong proteinic materials in the cytoplasm and
nucleolus. x 300. Fig. 10. Section in liver of a rat treated with CCl4
for 50 days showing moderate decrease in proteinic materials in most cells. x
300. Fig. 11. Section in liver of a rat treated with CCl4 for 130 days
showing a marked decrease of proteinic materials. x 300. Fig. 12.
Section in liver of a rat treated with CCl4 and thiola for 130 days showing
improvement in the amount and distribution of protein as compared with that
treated with CCl4 alone. x 300.
Fig. 13. Section in liver of a control rat stained by
Gomeri’s stain showing alkaline phosphatase activity. x 300. Fig. 14.
Section in liver of a rat treated with CCl4 for 50 days showing increase in
alkaline phosphatase activity. x 300. Fig. 15. Section in liver of a rat
treated with CCl4 for 90 days showing a high increase in alkaline phosphatase
activity in the nucleus of the hepatocytes. x 300. Fig. 16. Section in
liver of a rat treated with CCl4 and thiola for 90 days showing moderate
alkaline phosphatase activity. x 165.
DISCUSSION
Results
obtained in the present work indicated that CCl4 treatment induced some histochemical changes in the liver of
rats. These results had proceeded in an almost parallel manner to the
corresponding histopathological ones reported by Sakr et al. (1994). Treating
rats with CCl4 caused marked changes in the glycogen of hepatic cells. However,
there was a general tendency toward a decrease in the amount of glycogen, which
was marked in the later stages of treatment, especially in the cirrhotic liver.
The
effect of CCl4 on glycogen amount of liver cells was demonstrated by many
investigations. Lockard et al. (1983) reported that rats treated with CCl4
showed a temporary decrease in the percentage of hepatocytes with glycogen.
CCl4 was found to reduce the quantity of liver glycogen (Trinus et al. 1986;
Bernacchi et al. 1988; Kwon et al. 1989) and the quantity of blood glucose
(Dubal & Bais 1982). Rastogi & Rama (1990) showed that CCl4 brings
about a rise in cytosolic free calcium. It also depletes glycogen and
glucose-6-phosphatase level in the liver. Hickenbottom & Hornbrook (1971)
reported that depletion of hepatic glycogen, in response to CCl4 treatment, has
been linked to changes in activities of glycogen transferase and glycogen phosphorylase.
Treating
rats with CCl4 induced fatty infiltration in the liver starting at 50 days
after treatment. This result agree with Itoh (1988) who reported that fatty
liver was produced within 3 to 4 weeks after treatment with CCl4. Lockard et al.
(1983) found that in addition to morphological changes produced in the liver of
rats by CCl4 treatment there was an increase in the number of hepatocytes with
fatty infiltration. Kim & Labella (1987) reported that CCl4 caused
significant increase in hepatic lipid peroxidation. Levels of total lipid and
cholesterol of liver were also increased (Dwived et al. 1990). It was reported that fatty changes is a
common lesion in CCl4 treated liver and this term is used when there is
morphological evidence of excess intracytoplasmic fat (Kanase et al. 1986). The
mechanism of this change is complex and may be produced by many factors, e.g.
organelle injury at different subcellular loci, metabolic disorders, deficiency
of essential lipotropic factors, excessive mobilization of fat from
extrahepatic sources or varying combinations of any of these factors (Poli et
al. 1979).
Among
the findings in the present work is the noticeable decrease of protein content
in hepatic cells of CCl4 treated rats. This observation correlates with the
results of Lamb et al. (1984) who reported that CCl4 reduced the liver protein
content, and found significant decrease of 18% in total hepatic protein and 37%
in RNA content in rats given CCl4. Impairment of protein synthesis in CCl4
hepatotoxictiy was reported by several investigators. Treatment with CCl4
caused membrane lipid peroxidation, altered lipid metabolism and decreased
protein synthesis in the injured hepatocytes (Pappas et al. 1984; Boger et al.
1987; Hanma 1990).
Another
histochemical change prevailing in the present investigation is the increase in
alkaline phosphatase activity in liver of the treated animals. This result
agrees with Sherlock & Walshe
(1947) who described an increase of alkaline phosphatase activity in the degenerating
and necrotic liver cells. Stowell et al. (1950) found that in cirrhotic liver
induced by CCl4 in mice, alkaline phosphatase reaction was increased in the
nuclei and nucleoli of the hepatocytes, but the cytoplasm showed no activity.
Edress et al. (1980) reported that alkaline phosphatase activity was increased
in the liver of human with obstructive biliary cirrhosis, alcoholic cirrhosis
and bilharzial cirrhosis.
The
results observed in this work showed that treatment with thiola induced marked
improvement in the glycogen, total proteins and lipids of the liver of rats
treated with CCl4. However, a partial improvement was recorded in the alkaline
phosphatase activity. Similarly, Sakr et al. (1993) found that thiola
supplements to methotrexate-treated rats produced marked improvement of the
glycogen, total proteins and nucleic acids contents of the liver. The
efficiency of thiola and WR 2721 as chemical radioprotectors against gamma
radiation was investigated in the skin of mice by El-Bialy et al. (1992). They
found that polysaccharides decreased in the skin of irradiated animals. A
significant increase in polysaccharides was recorded in skin of animals
post-treatment with thiola or WR 2721. El-Aaser et al. (1985) reported that
thiola and WR 2721 were found to have a protective effect on protein synthesis
in the kidney, liver and intestine of albino rats. The levels of liver
triglyceride and serum lipid peroxide increased in rats treated with alcohol,
these increases were markedly suppressed in rats treated with alcohol followed
by thiola or glutathione (Harata et al. 1982). Torrielli et al. (1981)
mentioned that liver fatty infiltration was significantly depressed in animals
given glutathione, cysteine or thiola. Mathur et al. (1986) reported that the activity
of alkaline phosphatase was increased post-irradiation and after treatment with
thiola it showed lesser values.
Results
of the present work provide additional evidence for the protective action of
thiola. Thiola is capable of liberating SH group, which has an important
physiological function in the human body as an antitoxic agent, and in
enzymatic activity (Kumar 1985)
However, the multiple effects of sulfhydryl compounds make it difficult to
ascertain their exact detoxifying mechanism(s). It is speculated that the
protective effect of thiola, out of many alternative mechanisms, most likely is
due to its capacity as SH donor. This SH group may act directly through a
conjugative reaction with CCl4 or its metabolites (Sakr et al. 1994) leading to
inhibition of its hepatotoxic action.
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