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Radioprotective effect of silymarin against radiation induced hepatotoxicity
Pharmacological Research, Vol. 45, No. 6, 2002 doi:10.1006/phrs.2002.990, available online at http://www.idealibrary.com on
RADIOPROTECTIVE EFFECT OF SILYMARIN AGAINST RADIATION INDUCED HEPATOTOXICITY LAILA A. RAMADANa,∗ , HAMED M. ROUSHDYa , GAMAL M. ABU SENNAb , NOUR E. AMINa and OLA A. EL-DESHWa a Department
of Drug Radiation Research and Radiation Biology, National Centre For Radiation Research And Technology (NCRRT), Atomic Energy Authority, Cairo, Egypt, b Department of Physiology, Faculty of Science, Ain Shams University Accepted 16 April 2002
The radioprotective effect of silymarin using different modes of treatment against radiation (3 or 6 Gy) induced hepatotoxicity 1, 3 and 7 days post-irradiation was studied. Whole-body gammairradiation revealed an increase in serum alkaline phosphatase (AP) activity as well as liver glutathione reductase (GR) and glutathione peroxidase (GSH-PX) activities on the first postexposure day with respect to the control value. However, 3 days after radiation exposure, these parameters showed a significant decrease below the control level which persisted till the end of the experimental time except for serum AP activity that showed another increase on the seventh post-exposure day at 3 Gy dose of radiation. A gradual increase in serum alanine and aspartate aminotransferase (ALT&AST) as well as gamma glutamyl transpeptidase activities were observed due to irradiation throughout the experimental time. Administration of silymarin as single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses or as intravenous (i.v.) injection (50 mg kg−1 ), caused significant protection. Intravenous treatment showed the most pronounced protection. The protective effect of silymarin was attributed to its antioxidant and free radicals c 2002 Elsevier Science Ltd. All rights reserved. scavenging properties.
K EY WORDS : irradiation, silymarin, hepatotoxicity.
INTRODUCTION Flavonoids are phenolic compounds of plant origin in which antioxident properties have been attributed [1]. These properties seem to be due to their ability to scavenge free radicals and to chelate metal ions [1, 2]. Silymarin is a flavonoid complex consisting of silybin, which is the most active component, silydianin and silychristin [3]. Silymarin is frequently used in the treatment of liver diseases where it is capable of protecting liver cells directly by stabilizing the membrane permeability through inhibiting lipid peroxidation [4] and preventing liver glutathione depletion [5]. Several experimental studies have been done to assess whether silymarin can influence the course of radiation illness. Flemming [6] found that oral doses of silymarin to mice prevented the radiation-induced loss in body weight, increased the percentage of animal survival ∗ Corresponding author. Dr Laila A. Ramadan, Drug Radiation Research
Department, NCRRT, Atomic Energy Authority, P. O. Box 29, Nasr City, Cairo, Egypt. E-mail: laila [email protected] 1043–6618/02/$ - see front matter
and accelerated the recovery of the surviving mice. In regenerating liver of irradiated rats, silymarin partly alleviates the changes in nucleic acids, histones and some cytological indicators of the damage [7]. The aim of the present study is to assess the radioprotective effect of oral or intravenous (i.v.) administration of silymarin on hepatotoxicity as induced by two sublethal dose levels (3 and 6 Gy) of whole-body gammairradiation. MATERIALS AND METHODS
Animals Male adult Wistar albino rats weighing 120–170 g, were kept under standard conditions and were allowed free access to a standard diet and clean drinking water.
Irradiation Whole-body gamma-irradiation was performed at the National Centre for Radiation Research and Technology (NCRRT), Cairo, Egypt, using an AECL Gamma Cell-40 c 2002 Elsevier Science Ltd. All rights reserved.
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biological irradiator. Animals were irradiated at an acute single dose level of 3 or 6 Gy delivered at a dose rate of 0.012 Gy s−1 .
Drug Silymarin was purchased from Aldrich Chemical Co. The required dose was dissolved in 0.2 ml of propylene glycol in saline 75/25 v/v [8].
Experimental design Animals were divided into the following groups each of six rats: Control group. Animals were administrated propylene glycol in saline 75/25 v/v. Irradiated groups. Rats received a single dose of whole-body gamma-rays (3 or 6 Gy). Silymarin treated groups. Animals received either single (70 mg kg−1 ) [9], fractionated (490 mg kg−1 as 70 mg kg−1 daily for 7 days) oral doses or i.v. injection (50 mg kg−1 ) [8] of silymarin. Silymarin treated (single oral dose) & irradiated groups. Animals received a single oral dose (70 mg kg−1 ) 1 h before irradiation at 3 or 6 Gy. Silymarin treated (fractionated oral dose) & irradiated groups. Animals received fractionated oral doses (490 mg kg−1 as 70 mg kg−1 daily for 7 days) 1 h before irradiation at 3 or 6 Gy. Silymarin treated (i.v. injection) & irradiated groups. Animals were injected intravenously with (50 mg kg−1 ) silymarin 30 min before irradiation (3 or 6 Gy). Rats were examined 1, 3, and 7 days after irradiation or silymarin treatment. Blood samples were collected and serum samples were separated. The levels of serum alkaline phosphatase were determined according to the method of Kind and King [10]. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were estimated after the method of Reitman and Frankel [11]. Gamma glutamyl transpeptidase (GGTP) levels were determined following the method of Szasz [12]. Livers were removed and washed with ice-cold saline and blotted with pieces of filter paper, weighed and homogenized in ice-cold distilled water. In the liver homogenate, total protein, glutathione content, glutathione reductase and glutathione peroxidase activities were determined according to the methods of Lowery et al. [13], Beutler et al. [14], Manso and Wroblewski [15] and Lawrence and Burk [16], respectively. The biochemical data were statistically analysed and the Student’s t-test was used to evaluate the significance of differences observed.
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RESULTS
AP Whole-body gamma-irradiation at the dose level 3 Gy, resulted in a marked increase (33%) in serum alkaline phosphatase (AP) activity on the first day post-irradiation with respect to control value (Fig. 1). However, 3 days after radiation exposure, the AP activity decreased to 16% below the control level. This was followed by another increase on the seventh day post-irradiation. At the higher radiation dose level (6 Gy), the results showed a slight initial decrease in AP activity on the first day followed by a high significant decrease in the enzyme activity reaching 62% below the control value on the third day post-exposure. Treatments of rats with single (70 mg kg−1 ) or fractionated (490 mg kg−1 ) oral doses of silymarin, 1 h before irradiation at 3 Gy, showed almost complete restoration of the normal levels in the alkaline phosphatase activity throughout the experimentation period (Fig. 1). Administration of silymarin as single (70 mg kg−1 ) or fractionated (490 mg kg−1 ) oral doses, 1 h before irradiation at the 6 Gy dose level, showed a protective effect which was more pronounced on the first and seventh day post-exposure. On the third day the enzyme activity was still significantly lower than the normal control group (Fig. 1). Administration of silymarin as a single i.v. dose (50 mg kg−1 ) to male rats before irradiation (3 or 6 Gy) showed a significant protection against the radiation effect on serum AP activity that was extended till the end of the experiment (Fig. 1). AP activity was kept close to the normal value.
ALT and AST Gamma-irradiation (3 Gy) induced a significant increase in the activity levels of both serum ALT (130 and 200%) and AST (80 and 179%) on the third and seventh days post-irradiation, respectively (Figs 2 and 3). Exposure of animals to the 6 Gy dose level caused a highly significant increase in the serum activity levels reaching 159 and 718% of ALT and 65 and 447% of AST 3 and 7 days post-exposure, respectively (Figs 2 and 3). Treatment with silymarin as single or fractionated oral doses ameliorated the effect of radiation exposure (3 Gy) on ALT and AST activities. The enzyme activities dropped and became close to the normal control level on the first and third day post-exposure. On the seventh day, serum ALT and AST activities showed a significant elevation over that of the control value recording (78 and 112%) for ALT and (24 and 42%) for AST, when animals were treated with a single or fractionated oral dose 1 h before irradiation, respectively. Administration of silymarin as a single or fractionated oral dose, showed a significant amelioration of the 6 Gy radiation induced changes in ALT and AST enzyme activities as recorded on the first post-irradiation day. Although a significant protection was observed on the
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Fig. 1. Effect of single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses and single i.v. dose (50 mg kg−1 ) of silymarin before irradiation (3 or 6 Gy) on serum AP (IU l−1 ) of male rats on 1, 3 and 7 days post-irradiation. ∗ Significant difference from the control at P < 0.05. 0 Significant difference from irradiated group at P < 0.05. Silymarin group, irradiated group (2 Gy), silymarin + irradiated group (2 Gy), irradiated group (6 Gy), silymarin + irradiated group (6 Gy).
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Fig. 2. Effect of single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses and single i.v. dose (50 mg kg−1 ) of silymarin before irradiation (3 or 6 Gy) on serum ALT activity (U ml−1 ) of male rats on 1, 3 and 7 days post-irradiation. ∗ Significant difference from the control at P < 0.05. 0 Significant difference from irradiated group at P < 0.05. Silymarin group, irradiated group (2 Gy), silymarin + irradiated group (2 Gy), irradiated group (6 Gy), silymarin + irradiated group (6 Gy).
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Fig. 3. Effect of single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses and single i.v. dose (50 mg kg−1 ) of silymarin before irradiation (3 or 6 Gy) on serum AST activity (U ml −1 ) of male rats on 1, 3 and 7 days post-irradiation. ∗ Significant difference from the control at P < 0.05. 0 Significant difference from irradiated group at P < 0.05. Silymarin group, irradiated group (2 Gy), silymarin + irradiated group (2 Gy), irradiated group (6 Gy), silymarin + irradiated group (6 Gy).
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third and seventh day, the enzyme activities were still significantly higher than the control value (Figs 2 and 3). Intravenous injection with silymarin before irradiation of animals exerted a significant protection of ALT activity on the first and third days post-exposure to 3 and 6 Gy dose levels. It had only a slight effect on the change induced by the 6 Gy dose level in ALT activity as recorded on the seventh day post-exposure (Fig. 2). Silymarin caused a significant restoration of AST enzyme activity after exposure to the 3 Gy dose level. Although a significant protection was observed by silymarin pre-treatment after exposure to 6 Gy, the AST level still remained significantly higher than the control level reaching (14 and 42%) on the third and seventh days post-irradiation, respectively (Fig. 3).
GGTP Figure 4 shows the effect of whole-body gammairradiation (3 and 6 Gy) and/or silymarin on the gamma glutamyl transpeptidase (GGTP) activity. Gammairradiation caused a significant increase in GGTP activity which reached its maximum value (152 and 684%) on the seventh day after exposure to 3 or 6 Gy, respectively. A single oral dose (70 mg kg−1 ) of silymarin when administered before irradiation (3 Gy) induced a restoration of GGTP activity to the normal control value through the time intervals (Fig. 4). Repeated administration of silymarin (70 mg kg−1 daily for a week) induced a restoration of GGTP activity on the first day. After 3 and 7 days, the enzyme was still higher than the normal one (Fig. 4). Significant protection was shown after treatment with silymarin as single or fractionated oral doses before irradiation at the 6 Gy dose level, however, the enzyme activity was still significantly higher than the normal value through the time intervals. Administration of silymarin (i.v.) before irradiation protected animals against the effect of radiation (3 Gy) on GGTP activity, where the values became within the normal limits. A significant protection to GGTP activity was observed after administration of silymarin (i.v.) before the 6 Gy dose level of irradiation. However, the GGTP activity still remained significantly higher than the control group (by 35%) after 7 days post-irradiation (Fig. 4).
GSH Irradiation of male rats caused a significant decrease in glutathione content (GSH) compared with the control group (Fig. 5). The reduction was about 34 and 71% below the normal value after 3 days of exposure to 3 and 6 Gy dose level, respectively. A significant protection against radiation effect was observed after pre-treatment with silymarin. The protection was more pronounced when animals received an i.v. injection (50 mg kg−1 ) of silymarin (Fig. 5).
GR Whole-body irradiation of male rats resulted in an initial increase in the glutathione reductase (GR) activity
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(15 and 16%) on the first day followed by a significant decrease (17 and 17%) on the seventh day post exposure to 3 and 6 Gy, respectively (Fig. 6). Treatment of animals with an oral dose (single or fractionated) or intravenous dose of silymarin before irradiation at the 3 and 6 Gy dose level showed a remarkable radioprotective activity throughout the experimentation period. GR activity returned nearly to the normal value when animals were injected intravenously with silymarin (Fig. 6).
GSH-PX Figure 7 shows the effect of irradiation (3 and 6 Gy) and/or silymarin on glutathione peroxidase (GSH-PX) activity in liver homogenate. Irradiation at the 3 Gy dose level caused an initial increase in the GSH-PX activity on the first day post-irradiation. However, there were pronounced decreases (68 and 54%) on the third day postexposure to 3 and 6 Gy, respectively. A full restoration of GSH-PX activity was detected after pre-treatment with silymarin (as a single oral dose or as i.v. injection) before exposure to 3 Gy. When animals were treated with a fractionated oral dose, the GSHPX activity returned to the normal value on the first day, however, the activity was still significantly lower than the normal value on the third and seventh day, after the 3 Gy dose of irradiation. Although, silymarin protected male rats from the effect of radiation (6 Gy), the GSH-PX activity remained significantly lower than the normal value on the third and seventh days postirradiation (Fig. 7). DISCUSSION Ionizing radiation is known to induce various physiological, biochemical changes in humans and animals. The objective of this study was planned to detect to what extent silymarin can ameliorate the radiation-induced toxicity to liver tissue. In the present study, gamma-irradiation (3 Gy) induced an increase in serum AP activity on the first and seventh post-exposure day. However, after 3 days, there was a decrease in serum AP activity. This is in agreement with the results of Khamis and Roushdy [17]. On the other hand the lethal dose (6 Gy) produced a marked fall in AP activity after 1 and 3 days post-irradiation, which returned to a normal value after 7 days of exposure. These results are in agreement with those of Higman and Hanks [18]. The increase in AP activity in blood serum 1 day after exposure to 3 Gy, can be attributed to the possible release of this enzyme from different tissues associated with the obstruction of the blood stream to the liver [17]. The decrease in AP activity after irradiation may be due to the decrease in liver Mg2+ and Zn2+ , which are responsible for liver AP activation [17]. El-Deshw [19], observed a decrease in liver Zn content on the third postexposure day.
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Fig. 4. Effect of single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses and single i.v. dose (50 mg kg−1 ) of silymarin before irradiation (3 or 6 Gy) on serum GGTP activity (IU l−1 ) of male rats on 1, 3 and 7 days post-irradiation. ∗ Significant difference from the control at P < 0.05. 0 Significant difference from irradiated group at P < 0.05. Silymarin group, irradiated group (2 Gy), silymarin + irradiated group (2 Gy), irradiated group (6 Gy), silymarin + irradiated group (6 Gy).
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Fig. 5. Effect of single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses and single i.v. dose (50 mg kg−1 ) of silymarin before irradiation (3 or 6 Gy) on GSH content (µg mg−1 protein) of male rats on 1, 3 and 7 days post-irradiation. ∗ Significant difference from the control at P < 0.05. 0 Significant difference from irradiated group at P < 0.05. Silymarin group, irradiated group (2 Gy), silymarin + irradiated group (2 Gy), irradiated group (6 Gy), silymarin + irradiated group (6 Gy).
In the present study, the groups of rats irradiated at 3 or 6 Gy gamma-rays, showed a significant increase in the activities of both serum ALT and AST through the time intervals. This increase is in agreement with previous findings of El-Ghazaly and Ramadan [20]. The increase in serum aminotransferase activities by radiation may be due to the damage of cellular membranes of hepatocytes, which in turn leads to an increase in the permeability of cell membranes and facilitates the passage of cytoplasmic enzymes outside the cells leading to the increase in the aminotransferase activities in blood serum [17]. The results show that whole-body gamma-irradiation of rats at 3 or 6 Gy resulted in an increase in GGTP activity that reached its maximum value after 7 days post-exposure. These results are in agreement with those of Reva et al. [21], who observed an increase in liver and serum GGTP activity under all exposure doses (2.25, 5.66, 6.46, 7.75, 10.37 and 12.92 mc kg−1 ) of
X-irradiation. The increase in GGTP activity may be due to liver injury and increase in cell membrane oxidative damage [22], which may be due to increased free radical generation caused by irradiation [23]. Whole-body gamma-irradiation ( 3 or 6 Gy) induced a pronounced decrease in liver GSH content (Figs 1 and 2). GR activity increased on the first post-exposure day to 3 and 6 Gy, then decreased on the third and seventh day post-irradiation (Figs 3 and 4). While GSHPX increased only on the first post-exposure day to 3 Gy irradiation.The increase in GR and GSH-PX on the first day post-exposure is in agreement with the finding of Saada et al. [24]. Saada et al. [24] observed an increase in GSH-PX and GR on the first exposure day, while after 72 h, a significant decrease was recorded. They suggested that the GSH-PX activity increased to destroy the excess H2 O2 formed after exposure to radiation. The reaction is
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Fig. 6. Effect of single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses and single i.v. dose (50 mg kg−1 ) of silymarin before irradiation (3 or 6 Gy) on GR activity (U mg−1 protein min−1 ) of male rats on 1, 3 and 7 days post-irradiation. ∗ Significant difference from the control at P < 0.05. 0 Significant difference from irradiated group at P < 0.05. Silymarin group, irradiated group (2 Gy), silymarin + irradiated group (2 Gy), irradiated group (6 Gy), silymarin + irradiated group (6 Gy).
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Fig. 7. Effect of single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses and single i.v. dose (50 mg kg−1 ) of silymarin before irradiation (3 or 6 Gy) on GSH-PX activity (m mol mg−1 protein) of male rats on 1, 3 and 7 days post-irradiation. ∗ Significant difference from the control at P < 0.05. 0 Significant difference from irradiated group at P < 0.05. Silymarin group, irradiated group (2 Gy), silymarin + irradiated group (2 Gy), irradiated group (6 Gy), silymarin + irradiated group (6 Gy).
catalysed in the presence of reduced glutathione and the increase in glucose-6-phosphatase dehydrogenase that generates NADPH which is essential to reduce oxidized glutathione. This necessitates an increase in the activity of GR that uses NADPH to reduce glutathione with the generation of NADP. The net result is the destruction of H2 O2 . Kojima et al. [25] observed a significant increase in GSH-PX and GR activities in the brain and liver of mice 24 h after irradiation, which was consistent with the radical scavenging activity of these organs. On the other hand, the decrease in GSH content, GR and GSH-PX activities as recorded in the present study after 3 and 7 days post-irradiation are in agreement with
those recorded by Othman [23] and Saada et al. [24]. They recorded a significant depletion in the antioxidant system accompanied by enhancement of lipid peroxides in rats after whole-body gamma-irradiation. The present results show a remarkable increase in GGTP activity induced by radiation accompanied by a decrease in liver GSH content. Meister [26] observed that GGTP catalysed the hydrolysis of GSH to glutamate and cysteinyl glycine; Sarker et al. [27] stated that the decrease in reduced glutathione by irradiation could be due to oxidation of the sulphydryl group of GSH due to the decrease in GR and the enzyme which reduces the oxidized GSSG into a reduced form using NADPH
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as a source of reducing equivalent [28]. Sadani and Nadkarni [29] supposed that the significant decrease in tissue GSH might be a consequence of its enhanced utilization by GSH-PX and increased sinusoidal efflux of GSH to extra hepatic tissue for local defence. Administration of silymarin through any of the modes of treatment, for either of the doses of radiation used in present study, caused a generally significant protective and ameliorative effect against radiation induced hepatotoxicity. The protective action of silymarin is associated with its antioxidant properties, as it possibly acts as a free radicals scavenger [22]. Silymarin has been reported to act as a free radical scavenger, an inhibitor of lipid peroxidation and a plasma membrane stabilizer [22, 30]. It helps in cell division and regeneration of liver [31], acts as a preservative of liver GSH content and prevents lipid peroxidation [22]. Silymarin significantly restores the changes of enzyme activities (AP, ALT and AST) due to its antioxidant effect and its ability to act as a radical scavenger, thereby protecting membrane permeability [30]. In patients subjected to continuous ambulatory peritoneal dialysis (CAPD), treatment with milk thistle extracts silymarin and silibinin, alone or in combination with cysteine donors, activated the cellular thiol status of the peritoneal macrophage and restored the functional capabilities [32]. Silymarin possesses a hydroxyl group at C5 in addition to the carbonyl group at C4 , which may form a chelate with Fe2+ (4). This chelation can raise the activity to the level of most active scavengers, possibly by sitespecific scavenging [33]. The free hydroxyl groups at C5 and C7 on the silymarin structure may also favour the inhibition of lipid peroxidation by reacting with peroxy radicals [4]. This ability of silymarin leads to a significant increase in the cellular antioxidant defence machinery by ameliorating the deleterious effects of free radical reaction [33] and by the increase in GSH content, which is important in maintaining the ferrous state [26]. Based on the results of the present study on the effect of silymarin treatment on GSH of the liver cells, one can conclude that treatment with silymarin intravenously has a better ameliorative effect than other modes of treatment [6], since the greatest increase in GSH level occurred in the case of i.v. treatment. Meanwhile, the fractionated oral treatment with silymarin on the GSH level showed the least ameliorative effects, a case in agreement with Miguez et al. [34]. This may be partly due to the antioxidant effect of GSH content.
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butylated hydroxyanisole and butylated hydroxytoluene. Planta Medica 1986; 6: 438–40. Mira L, Silva M, Manso CF. Scavenging of reactive oxygen species by silibin dihemisuccinate. Biochem Pharmacol 1994; 48: 753–9. Valenzuela A, Monica A, Soledad V, Ricardo G. Selectivity of silymarin on the increase of the glutathione content in different tissues of the rats. Planta Medica 1989; 55: 420–2. Flemming K. Die therapeutische wirkung von silymarin bei rontgenbestrahlten Mausen. Arzneimittelforschung 1971; 21: 1373. Hakova H, Misurova E. The effect of flavobion on nucleic acids in tissues of rats irradiated with gamma rays. Bull Exp Biol Med 1992; 3: 275–7. Mereish KA, Bunner DL, Regland DR, Creasia DA. Protection against microcystin-L. R. induced hepatotoxicity by silymarin: biochemistry, histopathology and lethality. Pharmacol Res 1991; 8: 273–7. Gakova N, Mishurova E, Kropachova K. Effect of flavobion on nucleic acids in tissues of rats irradiated with gamma rays. Bull Exp Biol Med 1992; 133: 257–77. Kind DP, King EG. Practical clinical biochemistry 1. 5th edn, Varly H, Gownlock AH, Bell M, eds. London: William Heinemann Medical Books Ltd, 1954: 892. Reitman S, Frankel S. A colourimetric method for the determination of serum oxaloacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957; 28: 56–63. Szasz G. Method for the determination of serum gamma glutamyl transpeptidase. Clin Chem 1969; 15: 124. Lowry OH, Rosebrough AL, Farr AL, Randall RJ. Protein measurements with folin phenol reagent. J Biol Chem 1951; 193: 265. Beutler E, Duran O, Kelly BM. Improved method of determination of blood glutathione. J Lab Clin Med 1963; 61: 852–5. Manso C, Worblewski F. Glutathione reductase activity in blood and body fluids. J Clin Invest 1957; 37: 214–8. Lawrence RA, Burk RF. Glutathione peroxidase activity in selenium-deficient rat liver. Biochem Biophys Res Commun 1976; 71: 952–8. Khamis F, Roushdy HM. Synergistic radio-protective action of imidazole and serotonin on serum and liver enzymes in rats. Arab J Sci App 1991; 24: 19–36. Highman B, Hanks AR. Serum intestinl alkaline phosphatase in rats after 800 R whole-body or regional X-irradiation. Proc Soc Exp Biol Med 1970; 133: 1210–16. El-Deshw A GO. Study on carbohydrate metabolism and trace elements balance after radiation exposure and/or hormonal treatment MSc Thesis, Department of Zoology, Faculty of Science, Ain Shams University, Cairo, Egypt, 1994. El-Ghazaly MA, Ramadan LA. Alterations in some metabolic functions in male albino rats after irradiation and possible role of thiola. J Egypt Soc Toxicol 1996; 16: 105–9. Reva AD, Zhhivalynk OB, Lukyanenko AI, Egorova EG, Dovretskij AI. Influence of chronic low dose X-ray irradiation on gamma glutamyl transpeptidase activity in rat organs and tissues. Radiats Biol Radioecol 1995; 35: 869–72. Muriel P, Garciapina T, Alvarez VP, Mourelle M. Silymarin protects against paracetamol-induced lipid peroxidation and liver damage. J Appl Toxicol 1992; 12: 439–42. Othman AI. Radioprotective effect of mercapto-propionyl glycine and selenium on antioxidants and enzymes of heme biosynthesis in gamma-irradiated rats. J Egypt Ger Soc Zool 1998; 27: 43–55. Saada HN, Azab Kh Sh, Said OZ, Mohamed MA, Abbady MM. Role of genesing in the control of radiation induced injury in rat erythrocyte. J Egypt Ger Soc Zool 1999; 28: 191–9. Kojima S, Matuski O, Nomura T. Effect of small doses of gamma ray on glutathione synthesis in mouse. International Conference on Low Doses of Ionizing Radiation Biology Effect and Regulatory Control, Seville, Spain. 1997; 17–21. Meister A. Glutathione metabolism and its selective modification. J Biol Chem 1988; 263: 17205.
454 27. Sarker SR, Singh LR, Uniyal BP, Bhatnag VS. Radioprotective effect of liv. 52 and tissues reduced glutathione in experimental rats. Bombay-Hosp J 1988; 30: 41. 28. Floh L, Gunzler WA. Glutathione-dependent enzymatic oxidoreduction. In: Glutathione metabolism and function. Arias IM, Jakoby WB, eds. New York: Raven Press, 1976. 29. Sadani G, Nadkarni GD. Role of antioxidant enzyme defense in sparing rat hepatocytes from toxicity of ricin at low dose. Indian J Exp Biol 1994; 32: 354–5. 30. Hakova H, Misurova E, Kropacova R. The effect of silymarin on the concentration and total content of nucleic acids in tissues of continuously irradiated rats. Vet Med (Praha) 1996; 41: 113–9. 31. Srivastava SA, Rvind K, Srivastava S, Srivastava G, Patnaik K,
Pharmacological Research, Vol. 45, No. 6, 2002 Dhawan BN. Effect of picroliv and silymarin on liver regeneration in rat. Indian J Pharmacol 1994; 26: 19–22. 32. Tager M, Dietzmann J, Thiel U, Hinrich Neumann K, Ansorges S. Restoration of the cellular thiol status of peritoneal macrophages from CAPD patients by the flavonoids silibinin and silymarin. Free Radic Res 2001; 34: 137–51. 33. Abu Ghdeer A RM, Ali SE, Osman S AA, Abu Bedair FA, Abbady MM, El-Kady MR. Antagonistic role of silymarin against cardiotoxicity and impaired antioxidation induced by adriamycin and/or radiation exposure in albino rats. Pakistan J Biol Sci 2001; 4: 604–7. 34. Miguez MR, Anundi I, Luis A, Kai SP, Lindros O. Hepatoprotective mechanism of silymarin: no evidence for involvement of cytochrome P450 2 El. Chem Biol Interact 1994; 91: 51–63.
RADIOPROTECTIVE EFFECT OF SILYMARIN AGAINST RADIATION INDUCED HEPATOTOXICITY LAILA A. RAMADANa,∗ , HAMED M. ROUSHDYa , GAMAL M. ABU SENNAb , NOUR E. AMINa and OLA A. EL-DESHWa a Department
of Drug Radiation Research and Radiation Biology, National Centre For Radiation Research And Technology (NCRRT), Atomic Energy Authority, Cairo, Egypt, b Department of Physiology, Faculty of Science, Ain Shams University Accepted 16 April 2002
The radioprotective effect of silymarin using different modes of treatment against radiation (3 or 6 Gy) induced hepatotoxicity 1, 3 and 7 days post-irradiation was studied. Whole-body gammairradiation revealed an increase in serum alkaline phosphatase (AP) activity as well as liver glutathione reductase (GR) and glutathione peroxidase (GSH-PX) activities on the first postexposure day with respect to the control value. However, 3 days after radiation exposure, these parameters showed a significant decrease below the control level which persisted till the end of the experimental time except for serum AP activity that showed another increase on the seventh post-exposure day at 3 Gy dose of radiation. A gradual increase in serum alanine and aspartate aminotransferase (ALT&AST) as well as gamma glutamyl transpeptidase activities were observed due to irradiation throughout the experimental time. Administration of silymarin as single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses or as intravenous (i.v.) injection (50 mg kg−1 ), caused significant protection. Intravenous treatment showed the most pronounced protection. The protective effect of silymarin was attributed to its antioxidant and free radicals c 2002 Elsevier Science Ltd. All rights reserved. scavenging properties.
K EY WORDS : irradiation, silymarin, hepatotoxicity.
INTRODUCTION Flavonoids are phenolic compounds of plant origin in which antioxident properties have been attributed [1]. These properties seem to be due to their ability to scavenge free radicals and to chelate metal ions [1, 2]. Silymarin is a flavonoid complex consisting of silybin, which is the most active component, silydianin and silychristin [3]. Silymarin is frequently used in the treatment of liver diseases where it is capable of protecting liver cells directly by stabilizing the membrane permeability through inhibiting lipid peroxidation [4] and preventing liver glutathione depletion [5]. Several experimental studies have been done to assess whether silymarin can influence the course of radiation illness. Flemming [6] found that oral doses of silymarin to mice prevented the radiation-induced loss in body weight, increased the percentage of animal survival ∗ Corresponding author. Dr Laila A. Ramadan, Drug Radiation Research
Department, NCRRT, Atomic Energy Authority, P. O. Box 29, Nasr City, Cairo, Egypt. E-mail: laila [email protected] 1043–6618/02/$ - see front matter
and accelerated the recovery of the surviving mice. In regenerating liver of irradiated rats, silymarin partly alleviates the changes in nucleic acids, histones and some cytological indicators of the damage [7]. The aim of the present study is to assess the radioprotective effect of oral or intravenous (i.v.) administration of silymarin on hepatotoxicity as induced by two sublethal dose levels (3 and 6 Gy) of whole-body gammairradiation. MATERIALS AND METHODS
Animals Male adult Wistar albino rats weighing 120–170 g, were kept under standard conditions and were allowed free access to a standard diet and clean drinking water.
Irradiation Whole-body gamma-irradiation was performed at the National Centre for Radiation Research and Technology (NCRRT), Cairo, Egypt, using an AECL Gamma Cell-40 c 2002 Elsevier Science Ltd. All rights reserved.
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biological irradiator. Animals were irradiated at an acute single dose level of 3 or 6 Gy delivered at a dose rate of 0.012 Gy s−1 .
Drug Silymarin was purchased from Aldrich Chemical Co. The required dose was dissolved in 0.2 ml of propylene glycol in saline 75/25 v/v [8].
Experimental design Animals were divided into the following groups each of six rats: Control group. Animals were administrated propylene glycol in saline 75/25 v/v. Irradiated groups. Rats received a single dose of whole-body gamma-rays (3 or 6 Gy). Silymarin treated groups. Animals received either single (70 mg kg−1 ) [9], fractionated (490 mg kg−1 as 70 mg kg−1 daily for 7 days) oral doses or i.v. injection (50 mg kg−1 ) [8] of silymarin. Silymarin treated (single oral dose) & irradiated groups. Animals received a single oral dose (70 mg kg−1 ) 1 h before irradiation at 3 or 6 Gy. Silymarin treated (fractionated oral dose) & irradiated groups. Animals received fractionated oral doses (490 mg kg−1 as 70 mg kg−1 daily for 7 days) 1 h before irradiation at 3 or 6 Gy. Silymarin treated (i.v. injection) & irradiated groups. Animals were injected intravenously with (50 mg kg−1 ) silymarin 30 min before irradiation (3 or 6 Gy). Rats were examined 1, 3, and 7 days after irradiation or silymarin treatment. Blood samples were collected and serum samples were separated. The levels of serum alkaline phosphatase were determined according to the method of Kind and King [10]. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were estimated after the method of Reitman and Frankel [11]. Gamma glutamyl transpeptidase (GGTP) levels were determined following the method of Szasz [12]. Livers were removed and washed with ice-cold saline and blotted with pieces of filter paper, weighed and homogenized in ice-cold distilled water. In the liver homogenate, total protein, glutathione content, glutathione reductase and glutathione peroxidase activities were determined according to the methods of Lowery et al. [13], Beutler et al. [14], Manso and Wroblewski [15] and Lawrence and Burk [16], respectively. The biochemical data were statistically analysed and the Student’s t-test was used to evaluate the significance of differences observed.
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RESULTS
AP Whole-body gamma-irradiation at the dose level 3 Gy, resulted in a marked increase (33%) in serum alkaline phosphatase (AP) activity on the first day post-irradiation with respect to control value (Fig. 1). However, 3 days after radiation exposure, the AP activity decreased to 16% below the control level. This was followed by another increase on the seventh day post-irradiation. At the higher radiation dose level (6 Gy), the results showed a slight initial decrease in AP activity on the first day followed by a high significant decrease in the enzyme activity reaching 62% below the control value on the third day post-exposure. Treatments of rats with single (70 mg kg−1 ) or fractionated (490 mg kg−1 ) oral doses of silymarin, 1 h before irradiation at 3 Gy, showed almost complete restoration of the normal levels in the alkaline phosphatase activity throughout the experimentation period (Fig. 1). Administration of silymarin as single (70 mg kg−1 ) or fractionated (490 mg kg−1 ) oral doses, 1 h before irradiation at the 6 Gy dose level, showed a protective effect which was more pronounced on the first and seventh day post-exposure. On the third day the enzyme activity was still significantly lower than the normal control group (Fig. 1). Administration of silymarin as a single i.v. dose (50 mg kg−1 ) to male rats before irradiation (3 or 6 Gy) showed a significant protection against the radiation effect on serum AP activity that was extended till the end of the experiment (Fig. 1). AP activity was kept close to the normal value.
ALT and AST Gamma-irradiation (3 Gy) induced a significant increase in the activity levels of both serum ALT (130 and 200%) and AST (80 and 179%) on the third and seventh days post-irradiation, respectively (Figs 2 and 3). Exposure of animals to the 6 Gy dose level caused a highly significant increase in the serum activity levels reaching 159 and 718% of ALT and 65 and 447% of AST 3 and 7 days post-exposure, respectively (Figs 2 and 3). Treatment with silymarin as single or fractionated oral doses ameliorated the effect of radiation exposure (3 Gy) on ALT and AST activities. The enzyme activities dropped and became close to the normal control level on the first and third day post-exposure. On the seventh day, serum ALT and AST activities showed a significant elevation over that of the control value recording (78 and 112%) for ALT and (24 and 42%) for AST, when animals were treated with a single or fractionated oral dose 1 h before irradiation, respectively. Administration of silymarin as a single or fractionated oral dose, showed a significant amelioration of the 6 Gy radiation induced changes in ALT and AST enzyme activities as recorded on the first post-irradiation day. Although a significant protection was observed on the
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Fig. 1. Effect of single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses and single i.v. dose (50 mg kg−1 ) of silymarin before irradiation (3 or 6 Gy) on serum AP (IU l−1 ) of male rats on 1, 3 and 7 days post-irradiation. ∗ Significant difference from the control at P < 0.05. 0 Significant difference from irradiated group at P < 0.05. Silymarin group, irradiated group (2 Gy), silymarin + irradiated group (2 Gy), irradiated group (6 Gy), silymarin + irradiated group (6 Gy).
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Fig. 2. Effect of single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses and single i.v. dose (50 mg kg−1 ) of silymarin before irradiation (3 or 6 Gy) on serum ALT activity (U ml−1 ) of male rats on 1, 3 and 7 days post-irradiation. ∗ Significant difference from the control at P < 0.05. 0 Significant difference from irradiated group at P < 0.05. Silymarin group, irradiated group (2 Gy), silymarin + irradiated group (2 Gy), irradiated group (6 Gy), silymarin + irradiated group (6 Gy).
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Fig. 3. Effect of single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses and single i.v. dose (50 mg kg−1 ) of silymarin before irradiation (3 or 6 Gy) on serum AST activity (U ml −1 ) of male rats on 1, 3 and 7 days post-irradiation. ∗ Significant difference from the control at P < 0.05. 0 Significant difference from irradiated group at P < 0.05. Silymarin group, irradiated group (2 Gy), silymarin + irradiated group (2 Gy), irradiated group (6 Gy), silymarin + irradiated group (6 Gy).
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third and seventh day, the enzyme activities were still significantly higher than the control value (Figs 2 and 3). Intravenous injection with silymarin before irradiation of animals exerted a significant protection of ALT activity on the first and third days post-exposure to 3 and 6 Gy dose levels. It had only a slight effect on the change induced by the 6 Gy dose level in ALT activity as recorded on the seventh day post-exposure (Fig. 2). Silymarin caused a significant restoration of AST enzyme activity after exposure to the 3 Gy dose level. Although a significant protection was observed by silymarin pre-treatment after exposure to 6 Gy, the AST level still remained significantly higher than the control level reaching (14 and 42%) on the third and seventh days post-irradiation, respectively (Fig. 3).
GGTP Figure 4 shows the effect of whole-body gammairradiation (3 and 6 Gy) and/or silymarin on the gamma glutamyl transpeptidase (GGTP) activity. Gammairradiation caused a significant increase in GGTP activity which reached its maximum value (152 and 684%) on the seventh day after exposure to 3 or 6 Gy, respectively. A single oral dose (70 mg kg−1 ) of silymarin when administered before irradiation (3 Gy) induced a restoration of GGTP activity to the normal control value through the time intervals (Fig. 4). Repeated administration of silymarin (70 mg kg−1 daily for a week) induced a restoration of GGTP activity on the first day. After 3 and 7 days, the enzyme was still higher than the normal one (Fig. 4). Significant protection was shown after treatment with silymarin as single or fractionated oral doses before irradiation at the 6 Gy dose level, however, the enzyme activity was still significantly higher than the normal value through the time intervals. Administration of silymarin (i.v.) before irradiation protected animals against the effect of radiation (3 Gy) on GGTP activity, where the values became within the normal limits. A significant protection to GGTP activity was observed after administration of silymarin (i.v.) before the 6 Gy dose level of irradiation. However, the GGTP activity still remained significantly higher than the control group (by 35%) after 7 days post-irradiation (Fig. 4).
GSH Irradiation of male rats caused a significant decrease in glutathione content (GSH) compared with the control group (Fig. 5). The reduction was about 34 and 71% below the normal value after 3 days of exposure to 3 and 6 Gy dose level, respectively. A significant protection against radiation effect was observed after pre-treatment with silymarin. The protection was more pronounced when animals received an i.v. injection (50 mg kg−1 ) of silymarin (Fig. 5).
GR Whole-body irradiation of male rats resulted in an initial increase in the glutathione reductase (GR) activity
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(15 and 16%) on the first day followed by a significant decrease (17 and 17%) on the seventh day post exposure to 3 and 6 Gy, respectively (Fig. 6). Treatment of animals with an oral dose (single or fractionated) or intravenous dose of silymarin before irradiation at the 3 and 6 Gy dose level showed a remarkable radioprotective activity throughout the experimentation period. GR activity returned nearly to the normal value when animals were injected intravenously with silymarin (Fig. 6).
GSH-PX Figure 7 shows the effect of irradiation (3 and 6 Gy) and/or silymarin on glutathione peroxidase (GSH-PX) activity in liver homogenate. Irradiation at the 3 Gy dose level caused an initial increase in the GSH-PX activity on the first day post-irradiation. However, there were pronounced decreases (68 and 54%) on the third day postexposure to 3 and 6 Gy, respectively. A full restoration of GSH-PX activity was detected after pre-treatment with silymarin (as a single oral dose or as i.v. injection) before exposure to 3 Gy. When animals were treated with a fractionated oral dose, the GSHPX activity returned to the normal value on the first day, however, the activity was still significantly lower than the normal value on the third and seventh day, after the 3 Gy dose of irradiation. Although, silymarin protected male rats from the effect of radiation (6 Gy), the GSH-PX activity remained significantly lower than the normal value on the third and seventh days postirradiation (Fig. 7). DISCUSSION Ionizing radiation is known to induce various physiological, biochemical changes in humans and animals. The objective of this study was planned to detect to what extent silymarin can ameliorate the radiation-induced toxicity to liver tissue. In the present study, gamma-irradiation (3 Gy) induced an increase in serum AP activity on the first and seventh post-exposure day. However, after 3 days, there was a decrease in serum AP activity. This is in agreement with the results of Khamis and Roushdy [17]. On the other hand the lethal dose (6 Gy) produced a marked fall in AP activity after 1 and 3 days post-irradiation, which returned to a normal value after 7 days of exposure. These results are in agreement with those of Higman and Hanks [18]. The increase in AP activity in blood serum 1 day after exposure to 3 Gy, can be attributed to the possible release of this enzyme from different tissues associated with the obstruction of the blood stream to the liver [17]. The decrease in AP activity after irradiation may be due to the decrease in liver Mg2+ and Zn2+ , which are responsible for liver AP activation [17]. El-Deshw [19], observed a decrease in liver Zn content on the third postexposure day.
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Fig. 4. Effect of single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses and single i.v. dose (50 mg kg−1 ) of silymarin before irradiation (3 or 6 Gy) on serum GGTP activity (IU l−1 ) of male rats on 1, 3 and 7 days post-irradiation. ∗ Significant difference from the control at P < 0.05. 0 Significant difference from irradiated group at P < 0.05. Silymarin group, irradiated group (2 Gy), silymarin + irradiated group (2 Gy), irradiated group (6 Gy), silymarin + irradiated group (6 Gy).
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Fig. 5. Effect of single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses and single i.v. dose (50 mg kg−1 ) of silymarin before irradiation (3 or 6 Gy) on GSH content (µg mg−1 protein) of male rats on 1, 3 and 7 days post-irradiation. ∗ Significant difference from the control at P < 0.05. 0 Significant difference from irradiated group at P < 0.05. Silymarin group, irradiated group (2 Gy), silymarin + irradiated group (2 Gy), irradiated group (6 Gy), silymarin + irradiated group (6 Gy).
In the present study, the groups of rats irradiated at 3 or 6 Gy gamma-rays, showed a significant increase in the activities of both serum ALT and AST through the time intervals. This increase is in agreement with previous findings of El-Ghazaly and Ramadan [20]. The increase in serum aminotransferase activities by radiation may be due to the damage of cellular membranes of hepatocytes, which in turn leads to an increase in the permeability of cell membranes and facilitates the passage of cytoplasmic enzymes outside the cells leading to the increase in the aminotransferase activities in blood serum [17]. The results show that whole-body gamma-irradiation of rats at 3 or 6 Gy resulted in an increase in GGTP activity that reached its maximum value after 7 days post-exposure. These results are in agreement with those of Reva et al. [21], who observed an increase in liver and serum GGTP activity under all exposure doses (2.25, 5.66, 6.46, 7.75, 10.37 and 12.92 mc kg−1 ) of
X-irradiation. The increase in GGTP activity may be due to liver injury and increase in cell membrane oxidative damage [22], which may be due to increased free radical generation caused by irradiation [23]. Whole-body gamma-irradiation ( 3 or 6 Gy) induced a pronounced decrease in liver GSH content (Figs 1 and 2). GR activity increased on the first post-exposure day to 3 and 6 Gy, then decreased on the third and seventh day post-irradiation (Figs 3 and 4). While GSHPX increased only on the first post-exposure day to 3 Gy irradiation.The increase in GR and GSH-PX on the first day post-exposure is in agreement with the finding of Saada et al. [24]. Saada et al. [24] observed an increase in GSH-PX and GR on the first exposure day, while after 72 h, a significant decrease was recorded. They suggested that the GSH-PX activity increased to destroy the excess H2 O2 formed after exposure to radiation. The reaction is
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Fig. 6. Effect of single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses and single i.v. dose (50 mg kg−1 ) of silymarin before irradiation (3 or 6 Gy) on GR activity (U mg−1 protein min−1 ) of male rats on 1, 3 and 7 days post-irradiation. ∗ Significant difference from the control at P < 0.05. 0 Significant difference from irradiated group at P < 0.05. Silymarin group, irradiated group (2 Gy), silymarin + irradiated group (2 Gy), irradiated group (6 Gy), silymarin + irradiated group (6 Gy).
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Fig. 7. Effect of single (70 mg kg−1 ), fractionated (490 mg kg−1 ) oral doses and single i.v. dose (50 mg kg−1 ) of silymarin before irradiation (3 or 6 Gy) on GSH-PX activity (m mol mg−1 protein) of male rats on 1, 3 and 7 days post-irradiation. ∗ Significant difference from the control at P < 0.05. 0 Significant difference from irradiated group at P < 0.05. Silymarin group, irradiated group (2 Gy), silymarin + irradiated group (2 Gy), irradiated group (6 Gy), silymarin + irradiated group (6 Gy).
catalysed in the presence of reduced glutathione and the increase in glucose-6-phosphatase dehydrogenase that generates NADPH which is essential to reduce oxidized glutathione. This necessitates an increase in the activity of GR that uses NADPH to reduce glutathione with the generation of NADP. The net result is the destruction of H2 O2 . Kojima et al. [25] observed a significant increase in GSH-PX and GR activities in the brain and liver of mice 24 h after irradiation, which was consistent with the radical scavenging activity of these organs. On the other hand, the decrease in GSH content, GR and GSH-PX activities as recorded in the present study after 3 and 7 days post-irradiation are in agreement with
those recorded by Othman [23] and Saada et al. [24]. They recorded a significant depletion in the antioxidant system accompanied by enhancement of lipid peroxides in rats after whole-body gamma-irradiation. The present results show a remarkable increase in GGTP activity induced by radiation accompanied by a decrease in liver GSH content. Meister [26] observed that GGTP catalysed the hydrolysis of GSH to glutamate and cysteinyl glycine; Sarker et al. [27] stated that the decrease in reduced glutathione by irradiation could be due to oxidation of the sulphydryl group of GSH due to the decrease in GR and the enzyme which reduces the oxidized GSSG into a reduced form using NADPH
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as a source of reducing equivalent [28]. Sadani and Nadkarni [29] supposed that the significant decrease in tissue GSH might be a consequence of its enhanced utilization by GSH-PX and increased sinusoidal efflux of GSH to extra hepatic tissue for local defence. Administration of silymarin through any of the modes of treatment, for either of the doses of radiation used in present study, caused a generally significant protective and ameliorative effect against radiation induced hepatotoxicity. The protective action of silymarin is associated with its antioxidant properties, as it possibly acts as a free radicals scavenger [22]. Silymarin has been reported to act as a free radical scavenger, an inhibitor of lipid peroxidation and a plasma membrane stabilizer [22, 30]. It helps in cell division and regeneration of liver [31], acts as a preservative of liver GSH content and prevents lipid peroxidation [22]. Silymarin significantly restores the changes of enzyme activities (AP, ALT and AST) due to its antioxidant effect and its ability to act as a radical scavenger, thereby protecting membrane permeability [30]. In patients subjected to continuous ambulatory peritoneal dialysis (CAPD), treatment with milk thistle extracts silymarin and silibinin, alone or in combination with cysteine donors, activated the cellular thiol status of the peritoneal macrophage and restored the functional capabilities [32]. Silymarin possesses a hydroxyl group at C5 in addition to the carbonyl group at C4 , which may form a chelate with Fe2+ (4). This chelation can raise the activity to the level of most active scavengers, possibly by sitespecific scavenging [33]. The free hydroxyl groups at C5 and C7 on the silymarin structure may also favour the inhibition of lipid peroxidation by reacting with peroxy radicals [4]. This ability of silymarin leads to a significant increase in the cellular antioxidant defence machinery by ameliorating the deleterious effects of free radical reaction [33] and by the increase in GSH content, which is important in maintaining the ferrous state [26]. Based on the results of the present study on the effect of silymarin treatment on GSH of the liver cells, one can conclude that treatment with silymarin intravenously has a better ameliorative effect than other modes of treatment [6], since the greatest increase in GSH level occurred in the case of i.v. treatment. Meanwhile, the fractionated oral treatment with silymarin on the GSH level showed the least ameliorative effects, a case in agreement with Miguez et al. [34]. This may be partly due to the antioxidant effect of GSH content.
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