بحث منشور للدكتور وصفي ظاهر بعنوان THE PROTECTIVE EFFICACY OF VITAMINS (C AND E), SELENIUM AND
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THE PROTECTIVE EFFICACY OF VITAMINS (C AND E), SELENIUM AND
SILYMARIN SUPPLEMENTS AGAINST ALCOHOL TOXICITY
Shalan M.G.*, Abd Ali W. Dh.† , Shalan A.G. ‡
*Al-Arish Fac. of Education, Suez Canal Univ., Biological and Geological Sciences Dpt., North Sinai, Egypt.
†High Health Institute, Egdabia, Libya.
‡Port Said Fac. of Education, Suez Canal Univ., Biological and Geological Sciences Dpt., Port Said, Egypt
ABSTRACT: This study aimed at investigating the efficacy of vitamins (C and E), selenium and silymarin (an
antioxidant complex from Silybum marianum) supplementation in reducing toxic effects of ethanol on liver
weight and some blood parameters. Sixty male rabbits, individually housed in steel cages, were randomly
divided into three groups. The first was a control group, the second received balanced diet and daily 20% (v/
v) ethyl alcohol in their drinking water, the third received the same diet and 20% (v/v) ethanol in their drinking
water and treated with vitamin C (1 mg/100 g body weight, BW), vitamin E (1 mg/100 g BW), selenium (0.01 mg/
100 g BW) and silymarin (1 mg/100 g body weight) by gastric tube daily. Five animals per group were slaughtered
every two weeks and liver and blood samples were taken after 2, 4, 6 and 8 weeks of treatment. Ethanol
decreased body weight of rabbits and induced hepatomegally and apoptotic DNA fragmentation in hepatocytes.
Chronic alcohol consumption induced significant increases in serum glucose, triglycerides and cholesterol
levels whereas serum total protein content decreased. Significant increases in serum ALT, AST, ALP and LDH
activities were observed in ethanol-treated rabbits. The treatment of alcohol-abused animals with vitamins (C
and E), selenium and silymarin enhanced significant improvement in the biochemical, physiological and molecular
aspects indicating their protective effects against alcohol toxicity.
Key words: Rabbits; alcohol toxicity; vitamin C; vitamin E; selenium, silymarin.
W O R L D
RA B B I T
SCIENCE
INTRODUCTION
Alcohol consumption represents a large problem all over the world (Kumar and Clark, 2002). Alcohol
cannot be stored and obligatory oxidation must take place predominantly in the liver via alcohol
dehydrogenase. The production of potentially toxic acetaldehyde is enhanced and conversion to
acetate reduced (Sherlock and Dooley, 2002). The hydrogen produced replaces fatty acid as a fuel so
that fatty acids accumulate with consequent ketosis, triglyceridaemia, fatty liver and hyperlipidaemia
(Lieber, 1990). The conversion of alcohol to acetaldehyde also leads to inhibition of protein synthesis
(Bernal et al., 1992) and alter its metabolism (Apte et al., 2004). Ethanol was also reported to cause
many alterations in the activities of several enzymes under different nutritional conditions which
appeared to play a role in modulating this effect (Shalan, 1996). Ethanol was recorded to alter also
carbohydrate (Martin et al., 2004) and lipid (Ruf, 2004) metabolism.
Koyuturk et al. (2004) showed the protective effect of combination therapy with vitamins C and E,
and selenium on ethanol-induced duodenal mucosal injury. Marino et al. (2004) showed that vitamin
E protects against alcohol induced oxidative stress. The presence of selenium in combination with
vitamin E enhanced its activity in removing free radicals and prevented their formation (Saito et al.,
2003).
THE PROTECTIVE EFFICACY OF VITAMINS (C AND E), SELENIUM AND
SILYMARIN SUPPLEMENTS AGAINST ALCOHOL TOXICITY
Shalan M.G.*, Abd Ali W. Dh.† , Shalan A.G. ‡
*Al-Arish Fac. of Education, Suez Canal Univ., Biological and Geological Sciences Dpt., North Sinai, Egypt.
†High Health Institute, Egdabia, Libya.
‡Port Said Fac. of Education, Suez Canal Univ., Biological and Geological Sciences Dpt., Port Said, Egypt.
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SHALAN et al.
Silymarin is an antioxidant flavonoid complex derived from the herb milk thistle (Silybum marianum).
It was proved to have a protective effect against experimental hepatotoxicity by regulating the
actions of the ultrastructures of the liver cells and improving the activities of hepato-cellular enzymes
and bile production (Hagymasi et al., 2002).
Thus the main purpose of our study was to investigate the protective effect of combined
supplementation with vitamins (C and E), selenium, and silymarin against alcohol toxicity.
MATERIAL AND METHODS
Animals and experimental treatments
Sixty male laboratory New Zealand White rabbits 8 weeks old (Oryctolagus cuniculus) weighing
(1000 ± 100 g) were housed individually in steel cages where laboratory balanced diet and water were
initially provided under standard controlled conditions (25 ± 2oC and relative humidity of 25 ± 5%).
Animals were randomly divided into three groups. The first group was normal controls (20 animals).
The second was ethanol group (20 animals). Each animal received 30 ml 20% (v/v) ethanol/day as
drinking water (absolute ethanol purchased from Al-Gomhoria Chemical Co., Egypt) and fed with
balanced diet. The third one was alcohol + antioxidant group (20 animals). Each animal received 30 ml
20% (v/v) ethanol/day as drinking water, fed with balanced diet and supplemented with 1 mg vitamin
C/100g body weight (BW), 1 mg vitamin E/100 g BW, 1 mg silymarin/100 g BW and 0.01 mg selenium/
100g BW by gastric tube daily.
Vitamin E (DL-a-tocopherol) and selenium (sodium selenite) were obtained from Merk (Darmstadt,
Germany). Silymarin was commercially available from Sedico Pharmaceutical Co. (Cairo, Egypt).
Sample collection and biochemical analyses
Animals were weighed at the beginning of the experiment and before each sample collection. Every
2 weeks 5 animals per group were anaesthetized and rapidly dissected. Livers were weighed
immediately after dissection.
Samples were collected after 2, 4, 6 and 8 weeks of alcohol consumption. Blood samples were collected
from the inferior vena cava in glass centrifuge tubes, then centrifuged for 15 min at 1000 g in a cooled
centrifuge (4oC). Sera were separated and stored at –30oC in deep freezer till further biochemical
measurements. Serum total protein, glucose, triglycerides, cholesterol, ALT (alanine aminotransferase),
AST (aspartate aminotransferase), ALP (alkaline phosphatase) and LDH (lactate dehydrogenase)
concentrations were determined automatically using Integra 800 auto-analyzer (Liver Institute,
Menoufiya University, Egypt).
Preparation of tissues, gel preparation and electrophoresis of lysate tissue
After dissection, liver was removed, blotted on filter paper and weighed. Portions of 10 mg were
taken immediately for gel examinations and the remaining portions were stored at –30oC.
Gels were prepared with 1.8 % electophoretic grade agarose (BRL). The agarose was boiled in Trisborate
EDTA buffer (1 × TBE buffer; 89 mM tris, 89 mM boric acid, 2 mM EDTA, pH 8.3). The 0.5 μg/
ml ethidium bromide was added to gel at 40oC. Gels were poured and allowed to solidify at room
temperature for 1h before samples were loaded. The 10 mg hepatic tissue was squeezed and lysed in
200 μl lysing buffer (50 mM NaCl, 1mM Na2 EDTA, 0.5% SDS, pH 8.3) for at least 30 min. For
electophoretic pattern of nucleic acids of tissue lysate, 20 μl of lysate hepatic cells was loaded in
well, 5 μl 6 × loading buffer was added on the lysing tissue.
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DIETARY SUPPLEMENTATION AGAINST ALCOHOL TOXICITY
Electrophoresis was performed for 2 hours at 50 V in gel buffer (1 × TBE buffer). Gel was photographed
using a Polaroid camera while the DNA and RNA was visualized using a 312 nm UV transilluminator.
Nucleic acids extraction and molecular assessment for apoptosis
Nucleic acids extraction was based on salting out extraction method (Aljanabi and Martinez, 1997).
For apoptosis, the extracted DNA was gently resuspended with TE buffer supplemented with 5%
glycerol, gently pipetting, then the samples were mixed with 6 × loading buffer and loaded directly on
the gel (Hassab El-Nabi, 2004). The remained DNA was kept at –20oC for another loading. Apoptotic
bands appeared and located at 180, 360 and 540 bp.
Statistical analysis:
Data are presented as means ± s.d. in tables. Data were statistically analyzed by one-way analysis of
variance (Anova-Tukey test) using SPSS 10.1 software pakage. The P values < 0.05 were considered
significant.
RESULTS AND DISCUSSION
Effect of alcohol intake
The impact of prolonged alcohol consumption on the growth of experimental animals has been used
as a means to determine the overall toxicity of this compound (Wartburg and Popenberg, 1970). The
results of present study demonstrated significant decreases in body weight which reached its maximum
value (–20%) by the end of the 2nd week of alcohol intake (Table 1). The magnitude of depression in
body weight was attenuated thereafter by prolonged ethanol consumption (–15% after 8 weeks of
treatment). It is relevant in this respect to mention that chronic treatment of pregnant rats with
ethanol depressed feed and water consumption and weight gain (Abel, 1978). This depression in
weight gain of the alcohol-treated animals probably reflects the complication of the depressant
effects of ethanol on feed intake and impaired feed efficiency (Abel and Dintcheff, 1978).
The present data demonstrate that alcohol toxicity leads to not significant increase in the liver
weight and hepatosomatic index (Table 1). This alteration is accompanied with significant decreases
in total serum protein (Table 2). Such results lead to the suggestion that proteins are accumulated in
Table 1: Body weight, liver weight and hepatosomatic index during the trial
Control group Alcohol group Alcohol+antioxidant group
Rabbits, no. 5 5 5
Body weight (g): 2 week 1110±102b 890±143a 1010±82ab
4 week 1150±206 1046±177 1125±95
6 week 1270±186 1050±100 1150±98
8 week 1300±216b 1100±99a 1200±141ab
Liver weight (g): 2 week 59±14 60±12 60±14
4 week 60±15 64±14 61±10
6 week 61±15 68±16 61±11
8 week 61±16 72±16 62±14
Hepatosomatic index1 (%) 2 week 5.0±0.5 6.3±1.0 5.2±0.6
4 week 4.9±0.7 6.1±1.2 5.0±0.8
6 week 4.7±0.5 6.0±0.9 4.8±0.9
8 week 4.6±0.6 5.9±0.9 4.6±1.7
1 Hepatosomatic index: liver weight (g) / body weight (g) x 100). a, b: P<0.05
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SHALAN et al.
the liver instead of being transported to blood, in response to toxic effects of alcohol. It is of
significant importance, in this context, to mention the results of Shalan (1995) showing that hepatic
protein content markedly increased after alcohol intoxication.
Moreover, ethanol was reported to decrease the number of hepatic macrotubules which are the key
promoting secretion and intracellular transport of proteins and accordingly protein retention occurs
accompanied by accumulation of lipids due to the increase in fatty acid binding protein leading to
fatty liver and hepatomegally (Pignon et al., 1987), whereas other authors attributed hepatomegally,
produced in response to chronic alcohol consumption, to the increase in the size of the hepatic cells
and not to the increase of hepatocyte number (Israel et al., 1979). Cunnane et al., (1985) showed that
increased liver weights resulted from hepatic triglyceride accumulation after chronic alcohol abuse.
The decreased serum protein content might be interpreted in the light of the fact that alcohol inhibits
secretion of the newly synthesized glycoprotein and albumin by hepatocytes (Lakshman et al.,
1989).
Serum glucose was increased significantly at 6 and 8 weeks of alcohol consumption compared with
normal controls (Table 2). It was shown that alcohol induced hyperglycemia (Forsander et al., 1958)
that resulted from release of glucose from hepatic glycogen stores (Ammon and Estler, 1968). At the
same time decreased peripheral utilization of glucose with alcohol intake helps in rising blood glucose
level (Lochner et al., 1967); this may be associated with alcohol effect that decreases blood insulin
and rises glucagon level (Bucher and Weir, 1976).
Table 2: Serum total protein and glucose concentration.
Control group Alcohol group Alcohol+antioxidant group
Rabbits, no. 5 5 5
Total protein (g/l): 2 week 59.2±5.2 53.8±5.1 54.6±5.7
4 week 62.2±8.9b 50.9±5.2a 57.2±5.3ab
6 week 60.0±7.7b 44.8±4.7a 53.4±5.8b
8 week 63.6±5.4b 43.0±4.5a 56.4±6.1b
Glucose (mmol/l): 2 week 4.92±0.51 5.28±0.54 5.02±0.52
4 week 4.67±0.48 5.50±0.60 4.87±0.54
6 week 4.66±0.47b 5.61±0.64a 4.78±0.49b
8 week 5.02±0.52b 6.77±1.04a 5.89±0.69ab
a, b: P<0.05
Table 3: Serum triglycerides and cholesterol concentration
Control group Alcohol group Alcohol+antioxidant group
Rabbits, no. 5 5 5
Triglycerides (mmol/l): 2 week 1.07±0.16b 1.58±0.17a 1.44±0.15a
4 week 1.11±0.18b 1.61±0.18a 1.45±0.14a
6 week 1.16±0.14b 1.66±0.23a 1.54±0.16a
8 week 1.21±0.19b 1.64±0.20a 1.43±0.16ab
Triglycerides (mmol/l): 2 week 2.58±0.30 3.03±0.42 2.70±0.29
4 week 2.48±0.32b 3.07±0.30a 2.62±0.36ab
6 week 2.41±0.44 2.77±0.39 2.59±0.31
8 week 2.45±0.73 2.70±0.46 2.53±0.55
a, b: P<0.05
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DIETARY SUPPLEMENTATION AGAINST ALCOHOL TOXICITY
Ethanol induced significant increase in triglycerides concentrations at 2, 4, 6 and 8 weeks of treatment
compared with normal controls (Table 3). A significant increase in serum cholesterol content was
reported only at 4 weeks of treatment of rabbits with ethanol compared with normal controls (Table
3). Fatty liver is an important feature of alcohol abuse (Bernal et al., 1992). Alcohol decreased fatty
acids oxidation levels in the liver (Lieber, 1991) and that resulted in hepatic triglycerides accumulation
(Lamb et al., 1994). Baraona et al. (1973) indicated that rising serum triglycerides level related to
increased triglycerides synthesis resulted from increased fatty acids and alpha-glycerophosphate
availability during alcohol metabolism, and seemed that alcohol enhanced lipogenesis through
microsomes enhancement (Jenkins, 1984).
Results showed increased AST, ALT, ALP and LDH activities in response to alcohol administration
(Table 4). It was documented that alcohol causes modifications in the fluidity of membranes (Freund,
1979), permeability of these membranes (Ross, 1977), and their lipid composition (Hoek et al., 1988).
Therefore, alcohol may exert its effect through alteration of synthesis in the endoplasmic reticulum,
intracellular translocation and/or possibility of solubilization at the site of plasma membrane, hence
increasing the level of serum enzymes especially membrane-bound enzymes, like ALP, and cytosolic
enzymes, such as LDH and transaminases (ALT and AST). Elevated serum levels of ALP and LDH
were also observed by Yokoyame et al. (1993). Meanwhile, it was showed in previous studies that
chronic alcohol intoxication depresses hepatic enzyme activities, suggesting that elevated serum
enzyme activities might be induced as a result of enhanced release of hepatic enzymes into blood
stream due to liver cell injury (Singer and Kaplan, 1978).
Ethanol induced apoptotic DNA fragmentation of hepatocytes (Figure 1). Studies in mice and rats
revealed that both acute and chronic alcohol administration resulted in significant increases in
hepatocyte apoptosis (Goldin et al., 1993, Yacoub et al., 1995). Potential mechanisms of acute ethanolinduced
liver apoptosis include increased cytokine activity, Fas ligand (FasL) expression, and/or
Table 4: Serum ALT, AST, ALP and LDH activities (U/L)
Control group Alcohol group Alcohol+antioxidant group
Rabbits, no. 5 5 5
ALT activity (U/l): 2 week 62±9b 165±9a 62±7b
4 week 61±9b 170±10a 64±9b
6 week 61±6b 172±8a 65±8b
8 week 61±10b 170±6a 67±8b
AST activity (U/l): 2 week 183±20b 235±23a 187±16b
4 week 182±16b 233±20a 183±19b
6 week 181±20b 249±22a 182±16b
8 week 185±10b 243±17a 186±13b
ALP activity (U/l): 2 week 162±17b 200±24a 166±19b
4 week 163±11b 200±22a 164±13b
6 week 150±16b 202±19a 162±24b
8 week 162±13b 207±19a 165±13b
LDH activity (U/l): 2 week 5.12±0.63 6.76±0.95 6.11±0.58
4 week 5.18±0.56b 7.98±0.84a 6.05±0.64b
6 week 5.21±0.50b 9.17±0.94a 5.36±0.63b
8 week 5.24±0.b 10.96±0.94a 5.26±0.59b
a, b: P<0.05
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SHALAN et al.
oxidative stress (Kurose et al., 1997, Neuman et al., 2001). Ethanol-induced liver apoptosis involves
the activation of cysteine proteases or caspases (Deaciuc et al., 1999, Zhou et al., 2001).
Endonucleases and DNA fragmentation factors are activated during apoptosis, resulting in degradation
of chromatin DNA into internucleosomal units (Cohen and Duke, 1984, Liu et al., 1997).
Effect of antioxidant treatment
Supplementations with vitamins (C, E), selenium and silymarin reduced the effect of alcohol intake
on body weight (Table 1). There were significant differences in serum total protein and glucose
concentrations between alcohol and alcohol + antioxidants groups at 6 weeks of treatment only
(Table 2). Ethanol + antioxidants group showed increased triglycerides content significantly at 2, 4
and 6 weeks of treatment compared with normal controls, however not significant differences was
reported at 8 weeks (Table 3). Supplementations with antioxidants significantly reduced the effect of
alcohol intake on serum ALT, AST, ALP and LDH activities (Table 4).
Overall results indicated the highly protective effects of vitamins (C, E), selenium and silymarin
supplements against alcohol intoxication. Tawfik (1998) reported that vitamin E protects
polyunsaturated fatty acids from oxygen effects, and inhibits lipid peroxidation enhanced by ethanol
(Situnayake et al., 1990) by acting as a free radical scavenger. Vitamin E stabilizes biomembranes and
prevents lysis of phospholipids (Koning and Drijver, 1979). It was shown that vitamin E prevents
alterations in ionic permeability of cellular membrane occurred after alcohol intake (Aono et al., 1978,
Littleton, 1980).
Vitamin C acts as a free radical scavenger and reduces alcohol capacity for interacting with critical
molecules (Davidson, 1998). Blankenship et al. (1997) indicated that vitamin C protects cells from
undergoing apoptosis. Upasani et al. (2001) showed that the preventive activity of vitamins C and E
may related to their antioxidant efficacy that inhibits lipid peroxidation.
Figure 1: Apoptotic DNA fragmentation in liver of rabbits treated with ethanol and the protective role of
vitamins (C, E), selenium and silymarin supplements. Lanes 1 and 2: ethanol treated with antioxidant
supplements for 8 weeks; Lanes 3, 4 and 5: ethanol only intake for 8 weeks; Lanes 6 and 7: controls; M: 1 kp
ladder.
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DIETARY SUPPLEMENTATION AGAINST ALCOHOL TOXICITY
Silymarin is a natural mixture of antioxidants acting as free radical scavenger and preventing lipid
peroxidation (Soto et al., 1998). It was reported that silymarin improves liver function tests related to
hepatocellular necrosis and/or increases membrane permeability (Buzzelli et al., 1993). Ramadan et
al. (2002) reported that the protective effect of silymarin was attributed to its antioxidant and free
radical scavenging properties. It was suggested that silymarin modulates the cellular immunoresponse
and restores impaired liver function through its antioxidant capacity (Horvath et al., 2001). Feeding
of animals on silymarin-phospholipid complex normalized lipid metabolism and inhibited
atherosclerosis (Bialecka, 1997). The protective effect of silymarin may be attributed to its ability to
scavenge oxygen free radicals and to inhibite liver microsome lipid peroxidation (Mira et al., 1994).
It was recorded that alcohol intake may result in a decreased intake of other nutrients, maldigestion
and malnutrition (Lieber, 1988). Supplementation with antioxidants may repair the nutritional factors
that may be affected by ethanol toxicity. The combined antioxidant supplementation may alter ethanol
metabolism through their antioxidant capacities and thereby decreasing its toxic effects. Supplements
may exert their effects through rapid elimination of ethanol via bile or decreasing ethanol intestinal
absorption. Such suggestions need further studies to ensure the beneficial role of combined treatment,
through measuring blood ethanol concentrations and serum alcohol dehydrogenase concentrations.
In conclusion, the present study showed that treatment of alcoholic abused animals with vitamins (C
and E), selenium and silymarin supplements reduced toxic effects of ethanol.
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