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Research Article
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Biochemical and Pathological Study of Protective Effect of Vitamin E in Azathioprine-Induced Hepatotoxicity in Rat |
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B. Amouoghli Tabrizi,
D. Mohajeri,
G. Mousavi,
F. Farajzade,
A. Khodadadi,
S.B. Alizade
and
B. Reihani
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ABSTRACT
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In the present research, we decided to study about the protective effect of vitamin E against Azathiprine-induced toxicity. In this study, 40 male Wistar rats were divided to 4 groups (each group contains 10 rats). For the first group, as the control one, normal saline was given. The second and third groups received 20 mg kg-1 of vitamin E daily and for 7 days by Intra Muscular (IM) injection. The forth group, that had similar state with three others, normal saline was injected for 7 days. On the seventh day, both 3 and 4 group treated by 15 mg kg-1 Azathioprine as a single dose and Intra Peritoneal (IP) form. Two other groups only received the dissolvent of Azathioprine in the same dose and manner. Twenty four hours after Azathioprine injection, the animals after being weighted were anesthetized by ether and blood sample were taken via., the tail vein and pathological sample was got from liver. The samples were allowed to clot and then their serum was separated by centrifuge machine of 2500 rpm for 10 min. This study showed that Azathioprine-induced damage on liver in group 3 is less than group 4 and function of organ in group 3 is nearly same with control group. Results of this study demonstrated that vitamin E decrease Azathioprine-induced hepatotoxicity in rat. According to surveys that have done, the necrotic regions and hepatic cellular death in liver was so lesser in the group that treated together with Azathioprine and vitamin E than the group that treated only by Azathioprine and was so similar to control group. Generally, the pathological results of this study confirm the biochemical results.
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How
to cite this article:
B. Amouoghli Tabrizi, D. Mohajeri, G. Mousavi, F. Farajzade, A. Khodadadi, S.B. Alizade and B. Reihani, 2009. Biochemical and Pathological Study of Protective Effect of Vitamin E in Azathioprine-Induced Hepatotoxicity in Rat. Journal of Biological Sciences, 9: 339-344. DOI: 10.3923/jbs.2009.339.344 URL: https://scialert.net/abstract/?doi=jbs.2009.339.344
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INTRODUCTION
Azathioprine (AZA) is an immunosuppressive drug (Aarbakke
et al., 1997; Czaja, 1999) that use in medicine
and veterinary to remedy different disease like as Inflammatory Bowel Diseases
(IBD) (Ludwig and Stange, 1999; Pearson
et al., 2000), acute lymphoblast leukemia (Sood
et al., 2000), rheumatoid arthritis (Khodabakhshi,
2004), ulcerative colitis (Kader et al., 1998;
Sood et al., 2000) and etc., (Czaja,
1999) and synchronous use of this drug by corticosteroid is the best choice
to prevent the graft rejection (Di Landro et al.,
2000). Although, the Azathioprine is mostly use, but despite of its lymphocyte
suppressive effect in patient (Amin and Hamza, 2005),
there is evidence that it cause bone marrow toxicity, digestive system toxicity
an hepatotoxicity either (Lee and Farrell, 2001; Menor
et al., 2004). It has been cleared out that, by causing an oxidative
damage, this drug leads to liver toxicity (Watanabe et
al., 1979; Pearson et al., 2000; Lee
and Farrell, 2001). This drug produces free radical in the tissues, witch
is one of the most important toxicity factors in organs (Watanabe
et al., 1979; Kaplowitz, 1997). Watanabe
et al. (1979) confirmed that Azathioprine oral consumption increases
alkaline phosphatase (ALP), Gama Glutamyl Transferase (GGT) in liver. In addition,
from a pathological respect this drug cause necrosis in lobular center, proliferation
of mitochondria and endoplasmic reticule (Watanabe et
al., 1979). Sun et al. (1996) showed
that using Azathioprine, changes the rate of liver enzymes and increase Alanine
Amino Transferase (ALT), ALP and malonyl deltoid, but decrease glutamine rate.
These days, applying antioxidants in order to prevent disease such as cancer
or prostate disease, heart disease and etc. is recommended. Menor
et al. (2004) used N-acetyl-L-cysteine to decrease its side effects
in liver tissue and finally came to this end that this composition can play
a protecting role in liver tissue. Amin and Hamza (2005)
surveyed effects of saliva, hibiscus, rosmarinus against liver problems due
to using Azathioprine that indicated these plants can decrease the drugs
side effects in liver. Vitamin E is one of the fat soluble elements with delightful
antioxidant effect (Karakilcik et al., 2004),
results achieved from researchers studies convey that this drug plays
an important role in reducing and destroying free radicals (Karakilcik
et al., 2004; Ramirez Farias et al., 2008).
Karakilcik et al. (2004) demonstrated that using
vitamin E and C can decrease oxidative effects due to B1 aflatoxin in rabbits
liver. Andres and Cascales (2002) indicated that vitamin
E can reduce oxidative effects due to using Siklosporin A. Researchers in Mexico
University indicated that vitamin E and C are able to reduce oxidative damages
as a result of using ethanol in rats liver (Ramirez
Farias et al., 2008). As the rats hepatotoxicity is because
of oxidative Azathioprine drug and producing free radicals. The objective of
this study was to evaluate the effects of vitamin E against hepatotoxicity due
to Azathioprine prescription.
MATERIALS AND METHODS
Investigations using experimental animals were conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in the United States guidelines (United States National Institutes for Health publication No. 85-23, revised in 1985) and the Ethical Committee on Animal Care approved the protocol. Forty male Wistar rats that were apparently healthy have selected and divided into 4 equal groups. After body-weighting of each group by digital balance, all animals were kept in individual cages during the whole experimental period, under strict hygienic conditions and fed with standard ration for rat ad libitum, in lightning for 12 h and darkness for the same hours in 25°C to get used to the environment. Then 1.5 mL of blood sample was taken via the tail vein from the members of all groups and the samples were studied in a way that will describe as following.
For the first group, as the control one, normal saline was given. The second
and third groups received 20 mg kg-1 of vitamin E daily and for 7
days by IM injection. The forth group, that had similar state with three others,
normal saline was injected for 7 days. On the 7th day, both group 3 and 4 treated
by 15 mg kg-1 Azathioprine (Ramopharmin Pharmaceutical Lab-50 mg
per tablet) as a single dose and IP form. Two other groups only received the
dissolvent of Azathioprine in the same dose and manner. Twenty four hours after
Azathioprine injection, the animals after being weighted were anesthetized by
ether and blood sample were taken via the tail vein and pathological sample
was got from liver. The samples were allowed to clot and then their serum was
separated by centrifuge machine of 2500 rpm for 10 min. Biochemical parameters
including total bilirubin, ALT, AST, ALP, albumin, LDH, cholesterol, HDL, calcium,
phosphorous and triglyceride were measured by identification kit of biochemistry
and Biowave spectrophotometer apparatus. Tissue samples were fixed in formalin
10% and then pathology cope was produced of them. After Hem toxin and Eosin
staining, they were compared in terms of cellular damages such as degenerative
changes, cellular death and inflammation changes.
Statistical analysis: All biochemical results were expressed as Mean±SD. Significant differences among the groups were determined by one-way Analysis of Variance (ANOVA) followed by student t-test using the statistical analysis software (SPSS) Ver. 13, under Windows XP. Statistical significance was considered at p<0.05.
RESULTS
Biochemical study: Results from studying the animals average weight in several groups as initial weight average and final one are shown in Table 1. By surveying these results, one can see that animals of the control group and vitamin E and its receivers together with Azathioprine usage as an antioxidant against the drugs side effects, have meaningful statistical increment (p<0.05), but comparing the initial and final body weights averages in animals that received Azathioprine by itself, shows that meaningful statistics come down (p<0.05).
Table 2 shows the average of chemical parameters such as
total bilirubin, ALT, AST, ALP, albumin, LDH, cholesterol, HDL, calcium, phosphorus
and triglyceride in both control and Azathioprine receiving group and the group
got vitamin E and the group got vitamin E together with Azathioprine.
Table 1: |
Initial and final weight averages in several groups of animals |
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Dissimilar letter(s) in each row shows that there is a meaningful
difference (p<0.05) |
Table 2: |
Biochemical parameters in liver tissue |
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Data are expressed as Mean±SEM, N = 10. Dissimilar
letter(s) in each row shows that there is a meaningful difference (p<0.05) |
Average of serums bilirubin in Azathioprine group shows meaningful statistical increase than the control one (p<0.05), while average of serums bilirubin in group of Azathioprine with vitamin E does not indicate meaningful statistical difference with the control group, also in comparing group of vitamin E and the control group, there is no significant statistical differences.
In surveying ALT, AST and ALP enzymes, there is a significant statistical increase in Azathioprine group than the control one (p<0.05), while studies the average of serums AST and ALT enzymes in the control group with one receiving just vitamin E, there is no such a significant statistical differences.
Studying the average of serums biochemical parameters such as total protein, albumin, LDH, calcium, phosphorus and triglyceride among groups treated by Azathioprine, Azathioprine with vitamin E and only vitamin E with the control group, no significant statistical difference was seen.
Histopathological survey: Histological studying on liver tissue of groups under consideration was done. Histological parameters in this organ in the control group were normal. However, parameters which in group that has treated by Azathioprine showed some alternations, which can categorize them from mediate tissue damages to severe ones as following:
Necrotic and degenerated random points, vessel congestion and dilatation necrosis
and degeneration of preportal region, necrosis and destruction of vessels
epithelial, accumulation of Coupfer cells by phagocyting activity around the
preportal spaces, wide areas of degenerative necrosis in central parts of hepatic
lobes, lymphocytes and granulocytes influence in portal region, wide scattering
of degenerated necrosis and bleeding (Fig. 1-3).
Histopathological studies of liver tissue of the group that was treated by
vitamin E together with Azathioprine, there was no distinct change or damages
due to Azathioprine in liver tissue (Fig. 4). Studying the
hepatic damages in the group that treated by vitamin E together with Azathioprine,
indicated that, there were no microscopic distinct alterations of hepatocyte
damages except centrilobular necrotic regions, lack of slight arrangement and
usual arrangement of hepatocyts, slight dilatation of sinusoids, minor dilatation
of hepatic central vein and Coupfer cells regional accumulation (Fig.
4).
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Fig. 1: |
An image of hepatic tissue in animals of the group, treated
by Azathioprine. The arrows show wide areas of degenerative necrosis and
hemorrhage in central part hepatic lobes. Pay attention to abnormal dilatation
of central vein and its epithelial destruction (Hem toxin-Eosin staining,
magnification: 60x) |
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Fig. 2: |
Liver of animals in Azathioprine group, preportal severe necrosis
and portal vein destruction (thick arrow) and epithelium of biliary/cystic
duct (thin arrow). Atrophic remained preportal hepatocytes and as a result,
their sinusoid spaces are more dilated (Hem toxin-Eosin staining, magnification:
400x) |
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Fig. 3: |
A microscopic image of liver of animals in group that treated
by Azathioprine, shows wide areas of analyzed necrosis (thin arrow) and
hemorrhage (thick arrows) (Hem toxin-Eosin staining, magnification: 400x) |
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Fig. 4: |
A microscopic survey of liver of animals in group that treated
by Azathioprine and vitamin E together, shows centrilobular necrotic areas
(thick arrows) and Coupfer cells regional accumulation (thin arrows) (Hem
toxin-Eosin staining, magnification: 400x) |
DISCUSSION
Now a days, diseases such as cancers and auto-immune problems are common throughout
the world and increase continuously. In order to encounter against these troubles,
it involves using drugs that not only prevent disease progression, but has also
lessen side effects on natural function of cells and organs patients body.
Among these drugs, Azathioprine is one of the widely used of them in these days
(Lee and Farrell, 2001; Raza et
al., 2003). This drug is prescript in diseases such as IBD, acute lymphoblastic
leukemia, rheumatoid arthritis, ulcerative colitis, auto immune hepatitis, IgA
nephropathy, dermatologic disease and etc. It can prevent resynthesis of purine
bases and so preventing cellular replication by inhibiting the RNA and DNA synthesis.
The toxicity of this drug in different organs, such as bone marrow, liver, digestive
system and pancreas, following its usage is distinct. The toxicity of this drug
is because of producing free radicals in body. Surveying these results presented
that Azathioprine causing increment the amount of AST and ALT enzymes. ALT or
alanine amino transference is a cytoplasmic enzyme that has more value in diagnosis
of hepatic disease than other organs. Increment of this enzyme can be a sign
of hepatic failures following the drug prescription. Increment of ALT after
prescribing the Azathioprine was reported by Sun et al.
(1996). No significant changes were seen in ALT amount than the control
group at the time of using vitamin E along with the drug that can be due to
anti-oxidant and preservative effects of this drug against Azathioprine side
effects. AST or aspartate amino transference has various isoenzymes such as
muscular, hepatic, pancreatic and etc. so increasing the amount of this enzyme
can be seen in different disease. In prescribing Azathioprine, the amount of
this enzyme in serum, indicated a significant increase than the control group.
This amount, although has been mentioned in natural range in references, it
can be due to damages established in body by Azathioprine, but since lactate
dehydrogenize enzyme, which is under more consideration in muscular damages,
has no significant changes and increasing the amount of ALT enzyme together
with AST, therefore doubt about more hepatic failures. In using vitamin E together
by the drug, the amount of this enzyme did not present a significant difference
in compare with control group, which is because of these drugs anti-oxidant
effects against Azathioprine. The increment of this enzyme was reported by Sun
et al. (1996). By surveying the results achieved from present study,
its cleared that ALP enzyme has significant statistical differences in
Azathioprine treated group compare with three other groups. Since, the enzyme
has various isoenzymes, we can never relate its difference to hepatic problems,
but whereas the amount of Ca and P has no significant difference among the groups
of this research and attending that the hepatic and osseous ALP-isoenzymes are
the main ones, so we can say that its increasing is the consequence of
hepatic failures. By surveying the amount of serums ALP in the group that
was treated by Azathioprine together with vitamin E, there was no significant
statistical difference in quantities of this enzyme in compare with control
group that is the sign of its protective role against Azathioprine side effects.
Similar study was down by Amin and Hamza (2005) about
the effects of hibiscus, rosmarinus and salvia on Azathioprine-induced toxicity
in rats and the results is shown that these drugs can lessen toxicity effect
Azathioprine to somehow. The average of serums bilirubin in the group
that was treated only by Azathioprine has significant increment in compare with
control group. Since, this drug is metabolized and expelled in liver, can affect
on bodys antioxidant system, especially on hepatic tissue, increasing
bilirubin and other hepatic enzymes such as ALT and AST, perhaps can be due
to hepatic tissue damages. Using Azathioprine together with vitamin E can compensate
this increase, because this vitamin is a strong antioxidant and by simultaneous
consumption of it, the serums average bilirubin in these groups have no
significant statistical differences in compare with control group. The average
of serums cholesterol, LDH, total protein, albumin, HDL, Ca, P and triglyceride
have no significant changes in compare with control group in this research.
Histopathological results either indicate that simultaneous consumption of vitamin
E with Azathioprine, mainly reduces hepatic complications due to treating by
Azathioprine. According to surveys that have done, the necrotic regions and
hepatic cellular death in liver was so lesser in the group that treated together
with Azathioprine and vitamin E than the group that treated only by Azathioprine
and was so similar to control group. Generally the pathological results of this
study confirm the biochemical results.
CONCLUSION
First, since the toxicity of Azathioprine in different tissues has been approved, so we should try to use this drug as less as possible except in emergency occasions. Then, as this drug can affect bodys antioxidant system, its better to use vitamins and antioxidant drugs together with it. Finally, it is suggested that the amount of GSH and MDA that are considered part of antioxidant system in body, should be assessed after Azathioprine and vitamin E consumption.
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