Effect of silymarin on sodium fluoride-induced toxicity and oxidative stress in rat cardiac tissues

This study aim to evaluate the protective effect of silymarin on sodium fluoride-induced oxidative stress in rat cardiac tissues. Animals were pretreated with silymarin at 20 and 10 mg/kg prior to sodium fluoride consumption (600 ppm through drinking water). Vitamin C at 10 mg/kg was used as standard antioxidant. There was a significant increase in thiobarbituric acid reactive substances level (59.36 ± 2.19 nmol MDA eq/g tissue) along with a decrease in antioxidant enzymes activity (64.27 ± 1.98 U/g tissue for superoxide dismutase activity and 29.17 ± 1.01 μmol/min/mg protein for catalase activity) and reduced glutathione level (3.8 ± 0.15 μg/mg protein) in the tissues homogenates of the sodium fluoride-intoxicated rats. Silymarin administration to animals before sodium fluoride consumption modified the levels of biochemical parameters.


INTRODUCTION
Fluoride in water sources is known to induce both useful and harmful influence on human body.In intoxication, fluoride induces skeletal and dental fluorosis which causes damage to major tissues of the body such as the heart tissues (Sinha et al. 2008).Fluoride accumulation in soft tissues causes oxidative stress through inhibition of different enzymatic systems and increased generation of free radicals (Nabavi et al. 2012c).Fluorideinduced oxidative stress plays an important role in progression of a variety of cardiac disorders such as cardiac failure and ischemia (Sinha et al. 2008).Therefore, oxidant-antioxidant balance is an important mechanism in mitigation of the oxidative stress in cardiac tissues.Sinha et al. (2008) have shown that fluoride consumption causes myocardium injuries and dysfunction.Also, Nabavi et al. (2012c) have reported that fluoride increases oxidative stress through abnormal biochemical parameters in different tissues of rats.The free radical-induced oxidative stress is well known as an important mechanism of fluoride intoxication (Nabavi et al. 2012c).
Reactive oxygen species have an important role in cardiac failures.Knowing that reactive oxygen species play a crucial role in fluorideinduced cardiotoxicity and oxidative stress, studies have been carried out on the cardioprotective action of antioxidants against fluoride-induced toxicity in cardiac tissues (Sinha et al. 2008).Recent reports have determined the mitigative action of herbal antioxidants in some cardiac failures caused by free radicals and other reactive species (Nabavi et al. 2012c).Previously, the authors reported that natural antioxidants mitigated the oxidative injuries of fluoride in the cardiac tissues of rat (Nabavi et al. 2012b).
Silymarin (Figure 1) is a mixture of three benzopyranone compounds and well known as a herbal antioxidant constituent of Silybum marianum (milk thistle).Silybin (a), silydianin (b), and silychristin (c) (Figure 1) are major components of silymarin (Rašković et al. 2011).Its useful effects have been correlated to the antioxidant, antiproliferative and antiinflammatory actions on the basis of the specific signaling pathway regulation, transcription factor and gene expression (Rao and Viswanath 2007).
The purpose of the present work is to determine the ameliorative action of silymarin through its antioxidant effect in the cardiac tissues of rats exposed to sodium fluoride.

ANIMALS
Male Wistar rats were purchased from Pasteur Institute of Iran, Amol, and kept in an animal house of the Department of Biology, College of Sciences, University of Mazandaran, weighing between 200-250 g.The animals were allowed access to pellet diet and water ad libitum for two weeks prior to the experiments, for their acclimatization.All of the animals were kept in ventilated cages at 28-30°C, and 12/12 h light/dark cycles.All procedures were performed according to the norm of the ethical committee of University of Mazandaran Institutional Animal Care and Use Committee (Approval number: No.S-2009 UMZ).Rats were divided into the 5 groups containing 10 rats per groups.Animals of group I received the vehicle only (1 mg/kg/day) intraperitoneally for 1 week and served as normal.Silymarin at 10 and 20 mg/kg/day was administrated intraperitoneally to animals of groups II and III for 1 week prior to sodium fluoride intoxication (600 ppm) for next week (treated groups).Vitamin C (10 mg/kg/day), as a standard antioxidant, was administrated intraperitoneally to animals of group IV prior to sodium fluoride intoxication and served as positive controls.Finally, group V were pretreated with vehicle for same period prior to sodium fluoride intoxication (600 ppm) and served as toxin control.

ANIMAL ANESTHESIA
Rats were anesthetized with mixture of ketamine (60 mg/kg) and xylazine (5 mg/kg) and killed 24 hours after the last application.Animal heart was removed and washed three times by normal saline for complete blood removal.

HOMOGENATE PREPARATION
The whole heart tissues were weighed and homogenized in chilled potassium chloride (1.15%).The homogenates were centrifuged at 6,000 g for 15 min at 4°C.The supernatant was used for biochemical analysis.

PROTEIN DETERMINATION
The protein concentration was evaluated according to the method of Bradford (1976) using bovine serum albumin as the standard.

BIOCHEMICAL ANALYSIS
ESTIMATION OF LIPID PEROXIDATION ASSAY TBARS in heart tissues was evaluated by reaction with thiobarbituric acid (Shahidi et al. 2002).Briefly, reaction mixture (1.0 mL) was contained 0.1 M of phosphate buffer (0.58 mL; pH 7.4), tissue homogenate (0.2 mL), 100 mM of ascorbic acid (0.2 mL) and 100 mM of ferric chloride (0.02 mL).After incubation at 37°C for 60 min, reaction was stopped by addition of 10% of trichloroacetic acid (1.0 mL).Then, 1.0 mL of thiobarbituric acid (0.67%) was added to the reaction mixture and placed in boiling water bath for 20 min.The amount of thiobarbituric acid reactive substances formed was examined by recording the absorbance of reaction mixture at 535 nm using spectrophotometer against a reagent blank.
DETERMINATION OF SUPEROXIDE DISMUTASE ACTIVITY Superoxide dismutase activity was evaluated through the method of Nabavi et al. (2012a).Reaction mixtures included 1 mL of sodium carbonate (50 mM), 0.4 mL of nitroblue tetrazolium (25 μM) and 0.2 mL of hydroxylamine hydrochloride (0.1 mM).The homogenates (0.1 mL, 1:10, w/v) were added to the reaction mixtures.Finally, the absorbance of the reaction mixture was recorded at 560 nm.

DETERMINATION OF CATALASE ACTIVITY
Catalase activity was evaluated according to the method of Nabavi et al. (2012c).Briefly, a reaction mixture (3 mL) containing 50 mM of phosphate buffer (2.5 mL; pH 5.0), 5.9 mM of H 2 O 2 (0.4 mL) and heart homogenate (0.1 mL) was incubated for one min and absorbance of the sample was recorded at 240 nm.

DETERMINATION OF REDUCED GLUTATHIONE ACTIVITY
Reduced glutathione level was evaluated through the method of Ellman (1959).The homogenate sample (720 µL) was diluted and then trichloroacetic acid (5%) was added to the reaction mixture for precipitation of protein content in tissue homogenates.
Reaction was centrifuged at 10,000 g for 5 min and then the supernatant was taken.Ellman's reagent [5,5'-dithiobis (2-nitrobenzoic acid) solution] was added to the sample.Finally, the absorbance of the sample was recorded at 417 nm.The values are presented as means ± S.D. Differences between group means were estimated using a one-way analysis of variance followed by Duncan's multiple range tests.Results were considered statistically significant when p < 0.05.

RESULTS AND DISCUSSION
Figure 2 contain TBARS level (lipid peroxidation) of rat hearts.The results demonstrate that one week of fluoride intoxication increases TBARS level in rat heart (59.36 ± 2.19 nmol MDA eq/g tissue) in comparison with normal rat (43.51 ± 1.47 nmol MDA eq /g tissue).Also we found that pretreatment with Silymarin and vitamin C inhibit fluoride-induced lipid peroxidation levels (51.83 ± 2 nmol MDA eq /g tissue for 10 mg/kg of silymarin; 43.7 ± 1.99 nmol MDA eq /g tissue for 20 mg/kg of silymarin; and 44.52 ± 1.73 nmol MDA eq /g tissue for vitamin C).Also, we observed a similar pattern in catalase activity (Figure 4).Fluoride intoxication decreased enzyme activity (29.17 ± 1.01 µmol/min/mg protein) compared with the control group (45.36 ± 2.27 µmol/ min/mg protein).Pretreatment with silymarin and vitamin C preserved catalase activity in comparison to fluoride-intoxicated rat (34.12 ± 1.3 µmol/min/ mg protein for 10 mg/kg; 42.5 ± 1.6 µmol/min/mg protein for 20 mg/kg for silymarin; and 43.93 ± 2.18 U/mg protein for vitamin C).Results of superoxide dismutase activity have been summarized in Figure 3.We observed that pretreatment with silymarin and vitamin C diminished fluoride-induced abnormality in superoxide dismutase activity level of rat heart (86.88 ± 3.66 U/mg protein for 10 mg/kg, 107.74 ± 4.4 U/mg protein for 20 mg/kg and 106.25 ± 3.01 U/mg protein for vitamin C Vs. 64.27 ± 1.98 U/mg protein for fluoride intoxicated rat).The data of reduced glutathione levels in heart homogenates are summarized in Figure 5.The results show that fluoride exposure for one week significantly decreased reduced glutathione levels (3.8 ± 0.15 µg/mg protein).Silymarin administration evidently protects against fluorideinduced decreas in the level of reduced glutathione (4.98 ± 0.22 µg/mg protein for 10 mg/kg and 5.65 ± CARDIOPROTECTIVE ACTION OF SILYMARIN 0.26 µg/mg protein for 20 mg/kg).Treatment with Vitamin C showed mitigating activity similar to silymarin (5.3 ± 0.21 µg/mg protein).dismutase and catalase) and in the level of reduced glutathione in rat cardiac tissues (Sinha et al. 2008).Previously, it has been reported that some natural products with antioxidant ability have mitigative action (Nabavi et al. 2012c).
Natural antioxidants such as phenols and flavonoids show protective actions in different models of toxin-induced oxidative stress in cardiac tissues.Silymarin is a mixture of flavonoid components, and this natural antioxidant is isolated from Silybum marianum L. Gaertn (Milk thistle).Silymarin is composed primarily of three polyphenolic benzopyranone components, silybin, silydianin, and silychristin (Lee and Liu 2003).Silymarin has anti-inflammatory, anti-oxidative and anti-carcinogenic activities and so it has been tested in various in-vitro and in-vivo models of oxidative stress for protection of oxidative injuries in different tissues (Chtourou et al. 2010).
In this paper, we examine fluoride-induced oxidative injuries in cardiac tissues, which increase the production of reactive oxygen species.Furthermore, we examine the ameliorative actions of silymarin, a natural antioxidant, on fluorideinduced oxidative stress in rat cardiac tissues.In our present study, TBARS and reduced glutathione levels and activities of antioxidant enzymes (i.e., superoxide dismutase and catalase in rat cardiac tissues) were examined to evaluate the protective action.Antioxidants play a crucial role in mitigation and/or protection against oxidative stress induced by sodium fluoride through directly scavenging and/or inhibiting reactive oxygen species.Combining results of present study show that intraperitoneally administration of silymarin before sodium fluoride consumption prevents oxidative stress in rat cardiac tissues due to the It is well known that an increas in reactive oxygen species formation leads to oxidative stress, which has an important role in pathogenesis of different illnesses including cardiovascular, inflammation etc.
Among different types of reactive oxygen species, hydrogen peroxide plays a crucial role in oxidative damage.Furthermore, hydrogen peroxide, thiobarbituric acid reactive substances and the conjugated diene formation in the cardiac tissues were increased in acute heart failure, while antioxidant enzymes such as superoxide dismutase and catalase protected from these abnormalities.
It has been reported that fluoride consumption leads to excessive formation of free radical and other reactive substances and diminishes the activity of antioxidant enzymes (Nabavi et al. 2012c).Liu et al. (2003) demonstrated that fluoride consumption increased production of lipid peroxides, reactive nitrogen and oxygen species, leading to cellular damage, which affects the cellular systems.Fluoride caused oxidative stress in the cells by biphasic mechanisms; it acts as a free radical generator as well as an inhibitor of endogenous antioxidant enzyme systems (Ranjan et al. 2009).Sodium fluoride intoxication increases the level of lipid peroxidation along with a decrease in antioxidant enzymatic activities (superoxide antioxidant actions of this compound.According to the results of the present work, silymarin-rich foods can protect cardiac tissues from fluoride-induced oxidative stress.Future studies are needed for a better understanding of preventive and/or protective action mechanisms.These results can benefit future studies on silymarin for its applications in drug discovery and development.

Figure 2 -
Figure 2 -Effect of silymarin and vitamin C on TBARS level in heart's of sodium fluoride intoxicated rat. a p<0.001 versus normal rats.b p<0.01 versus normal group.c p>0.05 versus normal group.

Figure 3 -
Figure 3 -Effect of sodium fluoride intoxication on superoxide dismutase activity in the rat's heart.a p<0.001 versus normal rats.b p>0.05 versus normal group.

Figure 4 -
Figure 4 -Effect treatment on catalase levels in sodium fluoride intoxicated rat. a p<0.001 versus normal rats.b p>0.05 versus normal group.

Figure 5 -
Figure 5 -Protective effect of silymarin on sodium fluoride induced abnormality in reduced glutathione level in the heart homogenate of rat. a p<0.001 versus normal rats.b p>0.05 versus normal group.