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BJVM September 2014. Kafein Hepato Protektif
BJVM September 2014. Kafein Hepato Protektif
BJVM September 2014. Kafein Hepato Protektif
Original article
Summary
In the current study, we investigated the protective effects of the caffeine (Caff) on hepatic damage
induced by diethylnitrosamine (DEN) administration in rats. Animals were divided into four groups
with 10 animals in each group. Animals in group 1 were untreated (control). Rats in group 2 were
injected with a single dose of 200 mg/kg DEN, intraperitoneally (DEN group), those from group 3
were intraperitoneally injected with 100 mg/kg caffeine daily for four weeks (Caff group) and rats
from group 4 (DEN+Caff) received the same DEN dose as group 2 and daily caffeine treatments as
group 3. After 4 weeks, blood was collected for analysis of activities of aspartate aminotransferase
(AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). Liver specimens were taken
for histopathological examination. The single intraperitoneal administration of 200 mg/kg DEN to
rats resulted in significantly elevated levels of serum AST, ALT and ALP indicative of hepatocellular
damage. Histopathological examination revealed proliferation of stellate cells, necrosis, cell swelling
and karyomegaly in DEN and DEN+Caff groups, with lower intensity in the DEN+Caff group. The
results from the present study suggested that caffeine exhibited hepatoprotective properties against
diethylnitrosamine-induced hepatocellular damage in rats.
Key words: caffeine, diethylnitrosamine, liver, rat, toxicity
INTRODUCTION
sumption reduced increased serum aspar- rages (Liao et al., 2001). It is reported to
tate aminotransferase (AST) and alanine be metabolically activated by cytochrome
aminotransferase (ALT), which are mark- P450 enzymes to form reactive electro-
ers of liver injury, and decreased the risk philes which induce oxidative stress lead-
of chronic liver disease (Freedman et al., ing to cytotoxicity, mutagenicity and car-
2009). However, the major cellular and cinogenicity (Archer, 1989). Oxidative
molecular mechanisms of the hepatopro- stress is considered essential to DEN-
tective action of caffeine are poorly de- induced hepatotoxicity and the use of an-
fined (Okano et al., 2008). Some studies tioxidant agents reduced liver damage
attributed the hepatoprotective effect of (Vitaglione et al., 2004; Pradeep et al.,
caffeine in alcohol liver injury to its anti- 2007). Various plants and plant derived
inflammatory and antioxidant effects). products have been tested and found to be
Observations based on chemical studies effective against DEN-induced hepato-
showed that caffeine was able to scavenge carcinogenesis and hepatotoxicity (Poojari
reactive oxygen species (ROS), particu- et al., 2010; Pradeep et al., 2010; Zhang
larly the hydroxyl radical (OH), known to et al., 2012). Lee et al. (2007) reported
be generated in the body by irradiation that caffeine had a protective effect
with various electromagnetic frequencies against liver injury induced by carbon
such as exposure to UV, as well as by tetrachloride.
many ambient physiologic reactions in- In the light of these observations, it
volving oxygen utilisation (Shi et al., was decided to evaluate the efficacy of
1991; Stadler & Fay, 1995). Furthermore, caffeine against diethylnitrosamine in-
it was reported that caffeine decreased duced hepatocellular damage. Thus, the
tissue lipid peroxidation, protected mem- aim of the present study was to examine
branes from ROS damage and improved the antioxidant and protective effects of
oxidative stress control (Demirts et al., caffeine on hepatotoxicity induced by di-
2012). ethylnitrosamine in rats.
In food processing, nitrites are added
to smoked fish, pickled vegetables and
MATERIALS AND METHODS
cured meats to inhibit bacterial growth
and as colorants and flavour enhancers. In Animals and treatments
this process, some nitrites are converted to
nitrosamines due to the effect of heat and The study was approved by the Animal
gastric acid, making these foods the major Ethics Committee of the University of
dietary source of nitrosamines (Hill, 1988; Shahid Chamran University, Iran. Forty
Thirunavukkarasu & Sakthisekaran, 2003). female albino Wistar rats weighing
Nitrosamines are amongst the most potent 1805 g were kept in the laboratory under
toxins. N-nitrosodiethylamine (DEN) is an constant temperature (242 oC) for at least
N-nitroso alkyl compound described as an one week before and through the experi-
effective hepatotoxin in experimental ani- ment. The animals were fed a standard
mals, producing toxicity after repeated diet and water, available ad libitum and
administration (Jose et al., 1998). DEN is maintained in accordance with the guide-
found in a wide variety of foods such as lines prescribed by the Faculty of Science.
cheese, soybeans, smoked, salted and The experimental rats were divided
dried fish, cured meat and alcoholic beve- into four groups with 10 animals in each
group:
Group I (control): Animals were fed Fixed materials were embedded in paraf-
on the standard diet and served as con- fin and sections of 5 m thickness were
trol group. cut. Slides were stained with haematoxylin
Group II (DEN): Rats were injected and eosin for histopathological examina-
intraperitoneally with a single dose of tion.
diethylnitrosamine at 200 mg/kg
Statistical analysis
(Sigma Aldrich, USA) (Shaarawy et
al., 2009). The results were expressed as mean
Group III (Caff): Animals were intra- SEM of different groups. The differences
peritoneally injected with a daily dose between the mean values were evaluated
of 100 mg/kg caffeine (Sigma Aldrich, by ANOVA followed by Tukey-Kramer
USA) for four weeks. test. A value of P<0.05 was accepted as
Group IV(DEN+Caff): Rats were in- significant.
jected with DEN dose as group 2 and
received daily caffeine treatments as
RESULTS
group 3.
Biochemical assays Biochemical results
The treated and control animals were sac- Table 1 shows serum ALT, AST and ALP
rificed by decapitation after 4 weeks of concentrations in control and experimen-
treatment. For biochemical study, sera tal animals. Diethylnitrosamine (Group II)
were obtained by centrifugation of the induced hepatotoxicity is shown by a 2-
blood samples and stored at 20 oC until fold increase in the activity of AST and a
assayed. The serum activities of alanine 3-fold increase in ALT and ALP in the
aminotransferase (ALT), aspartate ami- serum of rats as compared to controls
notransferase (AST) and alkaline phos- (Group I). This increased activity of stu-
phatase (ALP) were determined using died liver enzymes after DEN challenge
commercially available kits (Pars Az- was significantly decreased on week 4 after
moon, Tehran, Iran). the treatment with caffeine (Group IV).
After sacrifice, the livers of rats were re- The histopathological examination of the
moved and fixed in 10% neutral formalin. liver in control or caffeine-treated rats
Table 1. Effect of caffeine on serum alanine aminotransferase (ALT), alkaline phosphatase (ALP)
and aspartate aminotransferase (AST) levels in serum during diethylnitrosamine induced hepatotox-
icity. Results are given as meanSEM (n=10). Group I: untreated rats; Group II: treated with a single
dose of 200 mg/kg DEN treated rats; Group III: treated with daily dose of 100 mg/kg caffeine over 4
weeks; Group IV: treated with DEN and caffeine at the same time.
A B
Fig. 3. Photomicrographs of liver specimens from rats treated with DEN + coffeine showing mild
necrosis and mild karyomegaly (A) and mild proliferation of hepatic stellate cells (B).
H & E, bar=20 m.
Table 2. Quantitative assessment of renal histological changes in rats with diethylnitrosamine in-
duced hepatotoxicity. Group I: untreated rats; Group II: treated with a single dose of 200 mg/kg DEN
treated rats; Group III: treated with daily dose of 100 mg/kg caffeine over 4 weeks; Group IV: treated
with DEN and Caff at the same time.
bird McGraw Hill, New York, pp. 673 Vitaglione, P., F. Morisco, N. Caporaso & V.
679. Fogliano, 2004. Dietary antioxidant com-
Shi, X., N. S. Dalal & A. C. Jain, 1991. Anti- pounds and liver health. Critical Reviews
oxidant behaviour of caffeine: Efficient in Food Science and Nutrition, 44, 575
scavenging of hydroxyl radicals. Food and 586.
Chemical Toxicology, 29, 16. Zhang, C. L., T. Zeng, X. L. Zhao, L. H. Yu,
Shaarawy, S. M., A. A. Tohamy, S. M. El- Z. P. Zhu & K. Q. Xie, 2012. Protective
gendy, M. Saad, Y. Zakaria, B. B. Abeer, effects of garlic oil on hepatocarcinoma
S. M. Maha, K. Emad & M. Khalid, 2009. induced by N-nitrosodiethylamine in rats.
Protective effects of garlic and silymarin International Journal of Biology Science,
on NDEA-induced rats hepatotoxicity. In- 8, 363374.
ternational Journal of Biology Science, 5,
549557.
Stadler, R. H. & L. B. Fay, 1995. Antioxida-
tive reactions of caffeine: Formation of 8- Paper received 23.09.2013; accepted for
oxocaffeine (1,3,7 trimethyl uric acid) in
publication 09.12.2013
coffee subjected to oxidative stress. Jour-
nal of Agricultural and Food Chemistry,
43, 13321338.
Thirunavukkarasu, C. & D. Sakthisekaran,
2003. Inuence of sodium selenite on gly-
coprotein contents in normal and N- Correspondence:
nitrosodiethylamine initiated and pheno-
barbital promoted rat liver tumors. Phar- Mojtaba Haghi Karamallah
macological Research, 48, 167173. Ph.D student of biochemistry,
International Campus,
Toyoki, Y., M. Sasaki, S. Narumi, S. Yoshi- Shahid Sadoughi University of
hara, T. Morita & M. Konn, 1998. Semi- Medical Sciences, Yaz, Iran,
quantitative evaluation of hepatic fibrosis Tel: +989166217343,
by measuring tissue hydroxyproline. E-mail: mojtabahaghi@ymail.com
Hepatogastroenterology, 45, 22612264.