- Email: [email protected]
Protective effect of resveratrol on sodium fluoride-induced oxidative stress, hepatotoxicity and neurotoxicity in rats
Food and Chemical Toxicology 70 (2014) 191–197
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Protective effect of resveratrol on sodium fluoride-induced oxidative stress, hepatotoxicity and neurotoxicity in rats Nurgul Atmaca a,⇑, Hasan Tarik Atmaca b, Ayse Kanici c, Tugce Anteplioglu b a
Kirikkale University, Faculty of Veterinary Medicine, Department of Physiology, Kirikkale, Turkey Kirikkale University, Faculty of Veterinary Medicine, Department of Pathology, Kirikkale, Turkey c Kafkas University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Kars, Turkey b
a r t i c l e
i n f o
Article history: Received 7 April 2014 Accepted 15 May 2014 Available online 22 May 2014 Keywords: Fluoride toxicity Oxidative stress Liver Brain Resveratrol Rats
a b s t r a c t Protective effect of resveratrol on sodium fluoride-induced oxidative stress, hepatotoxicity and neurotoxicity were studied in rats. A total of 28 Wistar albino male rats were used. Four study groups were randomly formed with seven animals in each. The groups were treated for 21 days with distilled water (control group), with water containing 100 ppm fluoride (fluoride group), with resveratrol (12.5 mg/kg i.p., resveratrol group), or with 100 ppm fluoride + 12.5 mg/kg resveratrol i.p. (fluoride + resveratrol group). At the end of the trial, blood samples were collected by cardiac puncture and tissue samples were taken simultaneously. The total antioxidant and oxidant status in plasma and tissues as well as plasma 8-hydroxydeoxyguanosine levels were measured. Histopathological analyses of rat liver and brain tissues were performed in all groups to identify any changes. In the fluoride group, the total oxidant levels increased in plasma, liver and brain and total antioxidant levels decreased, as did the plasma 8-hydroxy-deoxyguanosine levels. These changes were prevented by co-administration of resveratrol. In addition, fluoride-associated severe histopathological changes in brain and liver tissues were not observed in the fluoride + resveratrol group. Consequently, these data suggested that resveratrol had beneficial effects in alleviating fluoride-induced oxidative stress. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Fluoride, the most electronegative element, forms ionized fluorides with several other elements (Chlubek, 2003; Rzeuski et al., 1998), and is an important natural and industrial environmental pollutant (Whitford, 1983). Living organisms are exposed to fluoride via food, drinking water, fluoride additives, toothpastes, and professional administration of fluoride gel (Edmunds and Smedley, 1996). Fluorosis is a serious health condition caused by intense and prolonged exposure to inorganic fluoride, which is particularly prevalent in regions where fluoride is released into the air by the burning of fluoride-loaded coal, industrial production of phosphate fertilizers, and volcanic activity (USNRC, 1993), as well
Abbreviations: 8-OHDG, 8-hydroxydeoxyguanosine; MDA, malondialdehyde; NaF, sodium fluoride; TAS, total antioxidant status; TOS, total oxidant status. ⇑ Corresponding author. Address: Kirikkale University, Faculty of Veterinary Medicine, Department of Physiology, 71450, Yahsihan/Kirikkale, Turkey. Tel.: +90 3185374242; fax: +90 318 3573304. E-mail addresses: [email protected] (N. Atmaca), [email protected] (H.T. Atmaca), [email protected] (A. Kanici), [email protected] (T. Anteplioglu). http://dx.doi.org/10.1016/j.fct.2014.05.011 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
as in regions where fluoride-containing groundwater is used for drinking water (Wang et al., 2004). Increased production of reactive oxygen radicals, increased lipid peroxidation, and impaired antioxidant defense mechanisms are involved in the pathogenesis of fluoride toxicity (Vani and Reddy, 2000). Free radicals cause toxicity by attacking membrane phospholipids, leading to membrane injury through lipid peroxidation, depolarization of the mitochondrial membrane, and apoptosis (Barbier et al., 2010). Fluoride crosses the cell membrane (Carlson et al., 1960) and affects soft tissues, including the blood (Karadeniz and Altintas, 2008), brain (Shashi, 2003), and liver (Karaoz et al., 2003; Mittal and Flora, 2006). The liver has an active metabolism that renders it particularly sensitive to fluoride toxicity (Bouaziz et al., 2006; Guo et al., 2007). Many reports demonstrated that fluoride can induce lipid peroxidation and might cause change in the activity of some antioxidant enzymes in liver (Nabavi et al., 2012, 2013; Panneerselvam et al., 2013). Therefore, fluoride-induced oxidative stress and hepatotoxicity are associated with an imbalance in the oxidant/antioxidant systems of liver (Nabavi et al., 2012). Several studies performed on various animal species, including rats, sheep, and cattle, demonstrated that fluoride impaired liver function and
192
N. Atmaca et al. / Food and Chemical Toxicology 70 (2014) 191–197
metabolism, and caused histopathological changes (GruckaMamezar et al., 1997; Kapoor et al., 1993; Kessabi et al., 1986). Also, fluoride can accumulate in the brain (Geeraerts et al., 1986), which leads to abnormal behavioral patterns, impairment of neuronal and cerebrovascular integrity (Mullenix et al., 1995), and metabolic lesions (Vani and Reddy, 2000). Fluoride can also produce detrimental effects in brain tissue by inhibiting enzymes associated with energy production and transport, membrane transport, and synaptic transport (Vani and Reddy, 2000). Previous studies have also shown that fluoride increases lipid peroxidation and alters the levels of antioxidant enzymes in the brain (Flora et al., 2009; Shanthakumari et al., 2004) and liver (Guo et al., 2007; Hassan and Yousef, 2009; Mittal and Flora, 2006). Resveratrol is a compound obtained from the roots of the Polygonum cuspidatum plant used in traditional Eastern medicine for the treatment of fungal diseases, skin inflammation, and cardiovascular and liver diseases (Arichi et al., 1982). Resveratrol (trans3,5,40 -trihydroxystilbene) is a phenolic phytoalexin (Wang et al., 2002) that occurs naturally in various foods, including grapes, plums, cranberries, and peanuts. Its antioxidant effects have been demonstrated in the brain (Ates et al., 2007; Mokni et al., 2007; Sonmez et al., 2007), the liver (Dalaklioglu et al., 2013; TunaliAkbay et al., 2010), and the kidneys (Silan et al., 2007). Resveratrol is a free radical scavenger that increases the activity of several antioxidant enzymes (Gusman et al., 2001; Leonard et al., 2003). Although previous reports have demonstrated that the harmful effects of fluoride reduced because of concomitant intake of antioxidants, including black tea extract (Trivedi et al., 2012), a combination of vitamin E, methionine, and L-carnosine (Agha et al., 2012), Panax ginseng (Karadeniz and Altintas, 2008), pineal proteins, and melatonin (Bharti and Srivastava, 2009), quercetin (Nabavi et al., 2012), gallic acid isolated from Peltiphyllum peltatum (Nabavi et al., 2013), ferulic acid (Panneerselvam et al., 2013). To the best of our knowledge there is no published studies on the protective effects of resveratrol against fluoride toxicity. To address this lack of information, the objective of this study was to investigate the protective effect of resveratrol against oxidative stress caused by fluoride treatment in rats.
2.4. Tissue preparation The livers and brains were removed, washed, and perfused with normal saline to remove residual blood. The liver and brain tissues were homogenized (model TH 220, OMNI, Warrenton, VA, USA) 1:10 (w/v) in ice-cold 140 mM potassium chloride at pH 7.4. The homogenates were centrifuged at 3000 rpm for 10 min at 4 °C, and the supernatants were removed and stored at 80 °C until oxidative stress parameter analyses were performed. 2.5. Total antioxidant status assay Total antioxidant status (TAS) was measured using a commercially available kit from Rel Assay Diagnostics (Gaziantep, Turkey) (Erel, 2004). The method was based on the reduction of colored 2,20 -azino-bis(3-ethylbenzotiazoline-6-sulfonic acid) (ABTS) radical to a colorless reduced form by antioxidants present in the sample. Absorbance was measured spectrophotometrically at a wavelength of 660 nm. The method was calibrated using the vitamin E analog Trolox, and data were expressed as mmol Trolox equivalent (eq.) per liter (mmol Trolox eq./L). 2.6. Total oxidant status assay Total antioxidant status (TOS) was measured using a commercially available kit from Rel Assay Diagnostics (Erel, 2005). The method was based on the principle that the oxidants in the sample oxidized ferrous ions, previously bounded to a chelator, to ferric ions. In the acidic medium of the assay, these ferric ions formed a colored complex with a chromogen. The color intensity was measured spectrophotometrically at a wavelength of 530 nm. This assay was calibrated with hydrogen peroxide (H2O2), and the results were expressed as lmol H2O2 eq./L. 2.7. Determination of plasma 8-hydroxydeoxyguanosine The levels of 8-hydroxydeoxyguanosine (8-OHDG) were determined using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s protocol (Eastbiopharm, Hangzhou, China). The plasma samples were quantified by interpolation using the 8-OHDG standard curve assayed on each plate. The sensitivity limit of the ELISA system was 0.25 ng/mL of 8-OHDG. 2.8. Histopathological analysis Rat liver and brain tissues were fixed in 10% neutral formalin for 48–72 h. The tissues were then trimmed and processed for routine pathological examination. Then, they were embedded in paraffin wax and 4- to 5-lm-thick sections were cut. Hematoxylin and eosin staining was used for all tissue sections. Additionally, a 0.1% cresyl violet staining procedure was used to investigate neurodegenerative changes. Tissue slides were examined under a light microscope (Olympus BX51, Tokyo, Japan).
2. Materials and methods 2.9. Statistical analysis 2.1. Chemicals Sodium fluoride (NaF) was purchased from Merck (Darmstadt, Germany). trans-Resveratrol (>98% purity) was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). All other chemicals were obtained from Merck & Co., Inc. (White House Station, NJ, USA) or Sigma–Aldrich Corporation (St. Louis, MO, USA).
2.2. Animals and treatment Experiments were carried out using male Wistar albino rats weighing 180– 200 g that were fed a standard chow diet with water available ad libitum. Seven animals were housed in each plastic cage under a 12-h light/dark cycle (lights on at 08:00 am) at a constant temperature of 25 ± 2 °C with 42 ± 5% relative humidity. The study protocol was in accordance with the guidelines for animal research and approved by the Ethical Committee of the Kirikkale University (10/155). Twentyeight rats were randomly divided into 4 groups of 7 animals and treated as described below for 21 consecutive days. The control group received distilled water, the fluoride group received drinking water with 100 ppm fluoride, the resveratrol group received daily intraperitoneal (i.p.) administration of 12.5 mg/kg resveratrol, and the fluoride + resveratrol group received drinking water with 100 ppm fluoride and 12.5 mg/kg resveratrol i.p. daily.
2.3. Plasma collection After 21 days, all animals were sacrificed under light ether anesthesia and blood samples were collected into heparinized tubes by cardiac puncture. Plasma was separated by centrifugation at 3000 rpm for 10 min at 4 °C and used for the determination of total antioxidant status (TAS), total oxidant status (TOS), and 8-hydroxydeoxyguanosine (8-OHDG) levels.
Statistical analyses of data were performed using GraphPad Prism 3.0 software (GraphPad Software, San Diego, CA, USA). The data were expressed as mean ± standard error. One-way analysis of variance (ANOVA) was used to analyze the differences between groups. Post hoc comparisons were performed using Tukey’s multiple comparison test. P values less than 0.05 were considered as significant for all statistical calculations.
3. Results 3.1. Oxidative status in the plasma, liver, and brain There were statistically significant differences in TAS, TOS, and 8-OHDG levels between the fluoride group and the control group. Exposure to fluoride significantly elevated plasma TOS and 8-OHDG levels. Additionally, plasma TAS significantly decreased in the fluoride group, compared to those in the other groups. However, in the resveratrol and fluoride + resveratrol groups, these values were not significantly different from those observed in the control group (Table 1). In the fluoride group, TOS significantly increased in liver and brain tissues. In contrast, the TAS of these tissues in the fluoride group was significantly lower than that in the control group. However, TAS and TOS in the resveratrol group and in the fluoride + resveratrol group were not significantly different from those observed in the control group (Table 2). These findings indicated
193
N. Atmaca et al. / Food and Chemical Toxicology 70 (2014) 191–197 Table 1 Oxidative status and 8-OHDG levels in the study groups (n = 7). Parameters TAS (mmol Trolox eq./L) TOS (lmol H2O2 eq./L) 8-OHDG (ng/mL) a,b
Control
Fluoride a
1.14 ± 0.13 4.29 ± 0.65b 0.78 ± 0.09b
Fluoride + resveratrol b
0.76 ± 0.08 7.73 ± 0.38a 1.25 ± 0.08a
ab
0.93 ± 0.09 4.08 ± 1.02b 0.78 ± 0.09b
Resveratrol 1.04 ± 0.04a 3.79 ± 1.21b 0.82 ± 0.06b
The data were expressed as mean ± standard error. Data with a different superscript letter within the same row were significantly different from each other (p < 0.05).
Table 2 Oxidative status in the brain and liver of study groups (n = 7).
a,b
Parameters
Control
Fluoride
Fluoride + resveratrol
Resveratrol
Liver-TAS (mmol Trolox eq./L) Liver-TOS (lmol H2O2 eq./L) Brain-TAS (mmol Trolox eq./L) Brain-TOS (lmol H2O2 eq./L)
2.80 ± 0.23a 7.03 ± 0.48b 0.66 ± 0.03a 4.23 ± 0.14b
2.08 ± 0.14b 8.99 ± 0.38a 0.51 ± 0.01b 5.52 ± 0.22a
2.72 ± 0.10a 7.22 ± 0.33b 0.69 ± 0.03a 4.41 ± 0.29b
2.71 ± 0.09a 7.02 ± 0.48b 0.66 ± 0.04a 4.10 ± 0.26b
The data were expressed as mean ± standard error. Data with a different superscript letter within the same row were significantly different from each other (p < 0.05).
that resveratrol ameliorated the adverse effects of fluoride on TAS and TOS. 3.2. Histopathological assessment of liver and brain Brain histopathological analyses identified no changes in the cerebrum or cerebellum in control and resveratrol groups (Figs. 1 and 2). However, the fluoride group showed significant changes in the histology in various brain regions, and the hippocampus and cerebellum showed neurodegenerative changes. Of all the brain regions examined, the hippocampus showed the most noticeable changes in this study group. Neurons were shrunken and darkly stained, with small nuclei, and cell number decreased.
The Purkinje cells in the cerebellum were the most affected cell population in the fluoride group (Fig. 2c). The cerebrum and cerebellum of the fluoride + resveratrol group (Figs. 1d and 2d) showed structures similar to those in the control group. Histopathological examination of the liver showed normal structures in the control and resveratrol groups (Fig. 3a and b). However, the fluoride group showed pathological liver changes, including hydropic and vacuolar degeneration, and some necrosis. Slight liver congestion was also observed, as manifested by central vein and mild sinusoidal distension and occasional central hepatocyte degeneration (Fig. 3c). The fluoride + resveratrol group had liver tissue histology similar to that in the control group (Fig. 3d).
Fig. 1. Histological sections of the hippocampus of the study groups. (a) Control group hippocampus stained with hematoxylin and eosin (main panels) or cresyl violet (inset panels). Arrows indicate normal neurons and arrowheads indicate cresyl violet-stained neurons. The magnification was 200. (b) Resveratrol group hippocampus stained with hematoxylin and eosin (main panels) or cresyl violet (inset panels). Arrows indicate normal neurons and arrowheads indicate cresyl violet-stained neurons. The magnification was 200. (c) Fluoride group hippocampus stained with hematoxylin and eosin (main panels) or cresyl violet (inset panels). Note the degenerated neurons (arrows) and dark cresyl violet-stained shrunken neurons. The magnification was 200. (d) Fluoride + resveratrol group hippocampus stained with hematoxylin and eosin (main panels) or cresyl violet (inset panels). Note that the neurons appear normal and the number of degenerated neurons number was marginally less than that in the fluoride group (arrow). Some darkly stained shrunken neurons were observed using cresyl violet staining (arrow heads). The magnification was 200. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
194
N. Atmaca et al. / Food and Chemical Toxicology 70 (2014) 191–197
Fig. 2. Histological sections of the cerebellum of the study groups. (a) Control group cerebellum stained with cresyl violet. Normal neurons are indicated by arrows. The magnification was 200. (b) Resveratrol group cerebellum stained with cresyl violet. Normal neurons are indicated by arrows. The magnification was 200. (c) Flouride group cerebellum stained with cresyl violet. Darkly stained shrunken neurons are indicated by arrows. The magnification was 200. (d) Fluoride + resveratrol group cerebellum stained with cresyl violet. Note that the neurons had a normal appearance, and the number of degenerated (shrunken) neurons was marginally less than that observed in the fluoride group (arrows). The magnification was 200. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Histological sections of the liver of the study groups. (a) Control group liver stained with hematoxylin and eosin. The liver structure appeared normal. Vc = central vein. The magnification was 40. (b) Resveratrol group liver stained with hematoxylin and eosin. Liver structure appeared normal. Vc = central vein. The magnification was 40. (c) Fluoride group liver stained with hematoxylin and eosin. The asterisk indicates necrotic tissue, arrows indicate vacuolar degeneration, and hyperemia can be seen in sinusoids (arrow heads). Vc = central vein. The magnification was 200. (d) Fluoride + resveratrol group liver stained with hematoxylin and eosin. Some vacuolar degeneration was observed (arrow); however, in general, the tissue structure appeared normal. Vc = central vein. The magnification was 200.
4. Discussion 4.1. The resveratrol group Compared to the control group, the resveratrol group showed no statistically significant changes in oxidative stress parameters,
including plasma, liver, and brain TAS and TOS, and plasma 8-OHDG level. A previous study of the dose-dependent effects of resveratrol in the brain tissue of healthy rats reported decreased levels of malondialdehyde (MDA) in the brain, and increased antioxidant enzymes activities such as superoxide dismutase, catalase, and peroxidase (Mokni et al., 2007). The dose of resveratrol used in
N. Atmaca et al. / Food and Chemical Toxicology 70 (2014) 191–197
the present study was selected in line with this report, where the optimal dose of resveratrol for effects on antioxidant enzymes was 12.5 mg/kg (Mokni et al., 2007). The lack of any adverse oxidative stress-related effects of resveratrol in the present study was likely due to the strong antioxidant (Ignatowicz and BearDubowska, 2001), hepatoprotective (Kasdallah-Grissa et al., 2007; Tunali-Akbay et al., 2010), and neuroprotective (Bastianetto et al., 2000) effects of this substance. 4.2. The fluoride group Increased free radical production plays a major role in the pathogenesis of several diseases and toxicities (Halliwell, 1992). Studies have suggested that fluoride increases free radical production, and that these free radicals mediate its toxic effects on soft tissues (Rzeuski et al., 1998). In this study, the fluoride group showed increased TOS in plasma, liver, and brain tissue. This increase was statistically significant when compared to the control group. Furthermore, the fluoride group had significantly decreased TAS in plasma, and liver and brain tissues. These alterations indicated that a high dose of fluoride (100 ppm) administered for a 21-day period induced free radical formation that was not neutralized by endogenous antioxidant systems. The observed increase in TOS is also supportive of this finding. Reduced TAS might reflect the consumption of endogenous antioxidant molecules or a fluorideinduced inhibition of antioxidant enzyme activity. Fluoride has been shown to induce lipid peroxidation and decrease levels of several antioxidant molecules and enzyme activities in the blood (Hassan and Abdel-Aziz, 2010), the liver (Hassan and Yousef, 2009; Hassan and Abdel-Aziz, 2010; Nabavi et al., 2012, 2013; Panneerselvam et al., 2013), and the brain (Hassan and Abdel-Aziz, 2010). However, some studies have reported that fluoride did not affect antioxidant defense systems (Reddy et al., 2003). The current study and others have demonstrated that fluoride decreased total plasma antioxidant capacity in treated animals (Bouaziz et al., 2006; Hassan and Yousef, 2009; Hassan and AbdelAziz, 2010; Inkielewicz-Stepniak and Czarnowski, 2010). Fluoride has also been shown to produce marked increases in MDA and reactive products of thiobarbituric acid, which are indicators of lipid peroxidation caused by increased free radicals (Eraslan et al., 2007; Hassan and Yousef, 2009; Hassan and Abdel-Aziz, 2010; Inkielewicz-Stepniak and Czarnowski, 2010). In the present study, the increase in TOS, which indicated the total amount of reactive oxygen metabolites in plasma, and liver and brain tissues, and the decrease in TAS, suggested that the balance between oxidative and antioxidant systems was impaired in rats exposed to fluoride (Hassan and Abdel-Aziz, 2010), which resulted in oxidative stress. Additionally, fluoride administration increased levels of 8-OHDG, a marker of oxidative DNA injury (Loft and Poulsen, 1996). 8-OHDG is the main product of a hydroxyl radical attack on guanosine monophosphates (Harman, 1992), and is primarily removed from cells via DNA repair mechanisms (Burkhardt et al., 2001). The current study suggested that DNA damage occurred in fluoride-treated rats that DNA repair mechanisms were unable to counteract. In addition, histological analyses of the liver and the brain indicated that fluoride produced toxic effects on soft tissues. In the fluoride group, necrotic areas and hyperemia of the sinusoids were observed in liver tissue, as were degenerative neurons in the hippocampus and neural shrinkage in the cerebellum. The liver and the brain are particularly sensitive to free radical damage; liver tissue is highly metabolically active (Shashi and Thaper, 2000), and the multiple unsaturated fatty acid content in the brain produces an increased oxygen requirement (Watson, 1993). Fluoride has previously been reported to cause degenerative changes, ranging from balloon degeneration to hepatocellular necrosis and mononu-
195
clear cell infiltration, in the liver tissue of mice (Bouaziz et al., 2006) and rats (Karaoz et al., 2003; Panneerselvam et al., 2013). In addition, the brain has a low iron-binding capacity, as well as a significant level of iron ions that facilitate free radical production (Halliwell, 1992). Consistent with the findings of the present study, it has been reported that fluoride administration in rabbits caused chromatolysis and nuclear splitting in Purkinje cells, and shrunken, picnotic, and hyperchromatic neurons (Shashi, 2003). Fluoride administration in mice caused neurodegenerative changes in the cerebral hemisphere, cerebellum, and medulla oblongata, as well as chromatolysis, picnotic nuclei, fatty infiltration, and vacuolization (Trivedi et al., 2012). The neurodegenerative changes observed in the fluoride group suggested that fluoride crossed the blood– brain barrier and damaged neurons via oxidative changes. 4.3. The fluoride + resveratrol group Resveratrol is both a free radical scavenger and a potent antioxidant because of its ability to promote the activities of a variety of antioxidant enzymes (Alarcón de la Lastra et al., 2006). In addition, some investigators have indicated a potential hepatoprotective (Kasdallah-Grissa et al., 2007; Tunali-Akbay et al., 2010; Zhang et al., 2013) and neuroprotective (Ates et al., 2007; Sonmez et al., 2007; Lu et al., 2008) activities for resveratrol based on its beneficial effects in several liver and brain damage models. In this study, plasma TOS and TAS did not differ significantly between the fluoride + resveratrol group and the control group. However, plasma TOS in the fluoride + resveratrol group significantly decreased in comparison with that in the fluoride group, and TAS tended to increase, although not significantly. In addition, liver and brain tissue TOS and TAS were similar to those of the control group, but in both tissues, TOS was lower and TAS was higher, than those in the fluoride group. These plasma and liver, and brain tissue observations demonstrated the free radical scavenger and antioxidant effects of resveratrol. The effects of resveratrol on these parameters may be associated with its constituent compounds. Resveratrol is a polyphenolic compound and the most active of the stilbene phytoalexins (Kolouchova-Hanzlikova et al., 2004). The ability of the polyphenolic compounds to act as antioxidants depends on the redox properties of their phenolic hydroxyl groups and the potential for electron delocalization across the chemical structure (Ignatowicz and BearDubowska, 2001). Furthermore, the high hydrophilic and lipophilic content of resveratrol plays an important role in its efficacy in comparison to other antioxidants such as vitamins C and E (Kasdallah-Grissa et al., 2007). In this study, the antioxidant effects of resveratrol were considered to result from 3 mechanisms: first, the reduction of oxidative chain reactions of free oxygen species in competition with coenzyme Q; second, neutralization of superoxide radicals formed in the mitochondria; and third, prevention of lipid peroxidation by Fenton reaction products (Zini et al., 1999). The decrease observed in 8-OHDG levels, a biomarker of oxidative DNA injury, in the fluoride + resveratrol group compared to the fluoride group (Loft and Poulsen, 1996), also provided evidence for the antioxidant and free radical-scavenging activities of resveratrol. Similar to this study, resveratrol has been reported to play an effective role in preventing oxidative DNA injury in vivo (Guo et al., 2007; Tatlidede et al., 2009) and in vitro (Yan et al., 2012). Furthermore, the present study found that liver and brain tissues were generally normal in structure in the fluoride + resveratrol group, in contrast to the abnormal changes seen in the fluoride group. These results were consistent with reports of the strong antioxidant (Gulcin, 2010), hepatoprotective (Kasdallah-Grissa et al., 2007; Tunali-Akbay et al., 2010; Zhang et al., 2013), and neuroprotective (Ates et al., 2007; Mokni et al., 2007; Sonmez et al., 2007) activities of resveratrol.
196
N. Atmaca et al. / Food and Chemical Toxicology 70 (2014) 191–197
5. Conclusions These results have shown that oxidative stress participated in fluoride-induced toxicity, and resveratrol protected against fluoride-induced oxidative stress and tissue injury. Therefore, these results suggest that resveratrol inhibits the development of fluoride-induced hepatotoxicity and neurotoxicity in rats. Future studies will explore this effect in other model organisms, establish its dose–response relationship, and further examine the mechanisms involved. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version. Acknowledgment Financial support for this Project (Project No: 2011/44) was provided by the Kirikkale University Research Fund. References Agha, F.E., El-Badry, M.O., Hassan, D.A.A., Elraouf, A.A., 2012. Role of vitamin E combination with methionine and L-carnosine against sodium fluoride-induced hematological, biochemical, DNA damage, histological and immunohistochemical changes in pancreas of albino rats. Life Sci. J. 9, 1260–1275. Alarcón de la Lastra, C., Villegas, I., Martín, A.R., 2006. In: Aggarwald, B.B., Shishodia, S. (Eds.), Resveratrol in Health and Disease. CRC Press, Boca Raton, pp. 33–56. Arichi, H., Kimura, Y., Okuda, H., Baba, K., Kozawa, M., Arichi, S., 1982. Effects of stilbene components of the roots of Polygonum cuspidatum Sieb et Zucc on lipid metabolism. Chem. Pharm. Bull. 30, pp. 1776–1770. Ates, O., Cayli, S., Altinoz, E., Gurses, I., Yucel, N., Sener, M., Kocak, A., Yologlu, S., 2007. Neuroprotection by resveratrol against traumatic brain injury in rats. Mol. Cell. Biochem. 294, 137–144. Barbier, O., Arreola-Mendoza, L., Del Razo, L.M., 2010. Molecular mechanisms of fluoride toxicity. Chem-Biol. Interact. 188, 319–333. Bastianetto, S., Zheng, W.H., Quirion, R., 2000. Neuroprotective abilities of resveratrol and other red wine constituents against nitric oxide related toxicity in cultured hippocampal neurons. Br. J. Pharmacol. 131, 711–720. Bharti, V.K., Srivastava, R.S., 2009. Fluoride-induced oxidative stress in rat’s brain and its amelioration by Buffalo (Bubalus bubalis) pineal proteins and melatonin. Biol. Trace Elem. Res. 130, 131–140. Bouaziz, H., Ketata, S., Jammoussi, K., Boudawara, T., Ayedi, F., Ellouze, F., Zeghal, N., 2006. Effects of sodium fluoride on hepatic toxicity in adult mice and their suckling pups. Pestic. Biochem. Phys. 86, 124–130. Burkhardt, S., Reiter, R.J., Tan, D., 2001. DNA oxidatively damaged by chromium and H2O2 is protected by the antioxidants melatonin, N1-acetyl-N2-formayl-5methoxykynura mine, resveratrol and uric acid. Int. J. Biochem. Cell. 33, 775– 783. Carlson, C.H., Armstrong, W.D., Singer, L., 1960. Distribution and excretion of radio fluoride in the human. Proc. Soc. Exp. Biol. Med. 104, 235–239. Chlubek, D., 2003. Fluoride and oxidative stress. Fluoride 36, 217–228. Dalaklioglu, S., Genc, G.E., Aksoy, N.H., Akcit, F., Gumuslu, S., 2013. Resveratrol ameliorates methotrexate-induced hepatotoxicity in rats via inhibition of lipid peroxidation. Hum. Exp. Toxicol. 32, 662–667. Edmunds, W.M., Smedley, P.L., 1996. Groundwater geochemistry and health: an overview. Environ. Geochem. Health 113, 91–105. Eraslan, G., Kanbur, M., Silici, S., 2007. Evaluation of propolis effects on some biochemical parameters in rats treated with sodium fluoride. Pestic. Biochem. Phys. 88, 273–283. Erel, O., 2004. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin. Biochem. 37, 277–285. Erel, O., 2005. A new automated colorimetric method for measuring total oxidant status. Clin. Biochem. 38, 1103–1111. Flora, S.J.S., Mittal, M., Mishra, D., 2009. Co-exposure to arsenic and fluoride on oxidative stress, glutathione linked enzymes, biogenic amines and DNA damage in Mouse brain. J. Neurol. Sci. 285, 198–205. Geeraerts, F., Gijs, G., Finne, E., Crokaert, R., 1986. Kinetics of fluoride penetration in liver and brain. Fluoride 19, 108–112.
Grucka-Mamezar, E., Machoy, A., Tarnawski, R., Birkner, E., Mamezar, A., 1997. Influence of long term sodium fluoride administration on selected parameters of rat blood serum and liver function. Fluoride 30, 157–164. Gulcin, I., 2010. Antioxidant properties of resveratrol: a structure-activity insight. Innov. Food Sci. Emerg. 11, 210–218. Guo, L., Wang, L.H., Sun, B.S., Zhao, Y.Q., Dong, Y.X., Wu, C.F., 2007. Direct in vivo evidence of protective effects of grape seed procyanidin fractions and other antioxidants against ethanol-induced oxidative DNA damage in Mouse brain cells. Agric. Food Chem. 55, 5881–5891. Gusman, J., Malonne, H., Atassi, G., 2001. A reappraisal of the potential chemopreventive and chemotherapeutic properties of resveratrol. Carcinogenesis 22, 1111–1117. Halliwell, B., 1992. Reactive oxygen species and the central nervous system. J. Neurochem. 59, 1609–1623. Harman, D., 1992. Free radical theory of aging. Mutat. Res. 275, 257–266. Hassan, H.A., Abdel-Aziz, A.F., 2010. Evaluation of free radical-scavenging and antioxidant properties of black berry against fluoride toxicity in rats. Food Chem. Toxicol. 48, 1999–2004. Hassan, H.A., Yousef, M., 2009. Mitigating effects of antioxidant properties of black berry juice on sodium fluoride induced hepatotoxicity and oxidative stress in rats. Food Chem. Toxicol. 47, 2332–2337. Ignatowicz, E., Bear-Dubowska, W., 2001. Resveratrol, a natural chemopreventive agent against degenerative diseases. Pol. J. Pharmacol. 53, 557–569. Inkielewicz-Stepniak, I., Czarnowski, W., 2010. Oxidative stress parameters in rats exposed to fluoride and caffeine. Food Chem. Toxicol. 48, 1607–1611. Kapoor, V., Prasad, T., Bhatia, K.C., 1993. Effect of dietary fluorine on histopathological changes in calves. Fluoride 26, 105–110. Karadeniz, A., Altıntas, L., 2008. Effects of Panax Ginseng on fluoride-induced haematological pattern changes in mice. Fluoride 41, 67–71. Karaoz, E., Gulle, K., Mumcu, E.F., Gokcimen, A., Oncu, M., 2003. Deneysel kronik florozis olusßturulmusß 2. Kusßak sıçan böbrek ve karacig˘er dokularında yapısal deg˘isßiklikler. Turk. Klin. Tıp Bilim. 23, 129–134. Kasdallah-Grissa, A., Mornagui, B., Aouani, E., Hammami, M., El May, M., Gharbi, N., Kamoun, A., El-Fazaa, S., 2007. Resveratrol, a red wine polyphenol, attenuates ethanol-induced oxidative stress in rat liver. Life Sci. 80, 1033–1039. Kessabi, M., Hamliri, A., Braun, J.P., 1986. Experimental fluorosis in sheep: alleviating effects of aluminium. Vet. Hum. Toxicol. 28, 300–304. Kolouchova-Hanzlikova, I., Melzoch, K., Filip, V., Smidrkal, J., 2004. Rapid method for resveratrol determination by HPLC with electrochemical and UV detections in wines. Food Chem. 87, 151–158. Leonard, S., Xia, C., Jiang, B.H., Stinefelt, B., Klandorf, H., Harris, G.K., Shi, X., 2003. Resveratrol scavenges reactive oxygen species and effects radical-induced cellular responses. Biochem. Biophys. Res. Commun. 309, 1017–1026. Loft, S., Poulsen, H.E., 1996. Cancer risk and oxidative DNA damage in man. J. Mol. Med. 74, 297–312. Lu, K.T., Ko, M.C., Chen, B.Y., Huang, J.C., Hsieh, C.W., Lee, M.C., Chiou, R.Y.Y., Wung, B.S., Peng, C.H., Yang, Y.L., 2008. Neuroprotective effects of resveratrol on MPTPinduced neuron loss mediated by free radical scavenging. J. Agric. Food Chem. 56, 6910–6913. Mittal, M., Flora, S.J.S., 2006. Effects of individual and combined exposure to sodium arsenite and sodium fluoride on tissue oxidative stress, arsenic and fluoride levels in male mice. Chem-Biol. Interact. 162, 128–139. Mokni, M., Elkahoui, S., Limam, F., Amri, M., Aouani, E., 2007. Effect of resveratrol on antioxidant enzyme activities in the brain of healthy rat. Neurochem. Res. 32, 981–987. Mullenix, P.J., Enbesten, P.K., Schunior, A., Kernan, W.J., 1995. Neurotoxicity of sodium fluoride in rats. Neurotoxicol. Teratol. 17, 169–177. Nabavi, S.M., Nabavi, S.F., Eslami, S., Moghaddam, A.H., 2012. In vivo protective effects of quercetin against sodium fluoride-induced oxidative stress in the hepatic tissue. Food Chem. 132, 931–935. Nabavi, S.F., Nabavi, S.M., Habtemariam, S., Moghaddam, A.H., Sureda, A., Jafari, M., Latifi, A.M., 2013. Hepatoprotective effect of gallic acid isolated from Peltiphyllum peltatum against sodium fluoride-induced oxidative stress. Ind. Crops Prod. 44, 50–55. Panneerselvam, L., Subbiah, K., Arumugam, A., Senapathy, J.G., 2013. Ferulic acid modulates fluoride-induced oxidative hepatotoxicity in male Wistar rats. Biol. Trace Elem. Res. 151, 85–91. Reddy, G.B., Khandare, A.L., Reddy, P., Rao, G.S., Balakrishna, N., Srivalli, I., 2003. Antioxidant defence system and lipid peroxidation in patients with skeletal fluorosis and fluoride-intoxicated rabbits. Toxicol. Sci. 72, 363–368. Rzeuski, R., Chlubek, D., Machoy, Z., 1998. Interactions between fluoride and biological free radical reactions. Fluoride 31, 43–45. Shanthakumari, D., Sirinivasalu, S., Subramanian, S., 2004. Effect of fluoride intoxication on lipid peroxidation and antioxidant status in experimental rats. Toxicology 204, 219–228. Shashi, A., 2003. Histopathological investigation of fluoride-induced neurotoxicity in rabbits. Fluoride 36, 95–105. Shashi, A., Thaper, S.P., 2000. Histopathology of fluoride-induced hepatotoxicity in rabbits. Fluoride. 34, 34–42. Silan, C., Uzun, O., Comunog˘lu, N.U., Golcen, S., Bedirhan, S., Cengiz, M., 2007. Gentamicin-induced nephrotoxicity in rats ameliorated and healing effects of resveratrol. Biol. Pharm. Bull. 30, 79–83. Sonmez, U., Sonmez, A., Erbil, G., Tekmen, I., Baykara, B., 2007. Neuroprotective effects of resveratrol against traumatic brain injury in immature rats. Neurosci. Lett. 420, 133–137.
N. Atmaca et al. / Food and Chemical Toxicology 70 (2014) 191–197 Tatlidede, E., Sehirli, O., Velioglu-Ogunç, A., Cetinel, S., Yegen, B.C., Yarat, A., Süleymanoglu, S., Sener, G., 2009. Resveratrol treatment protects against doxorubucin-induced cardiotoxicity by alleviating oxidative damage. Free Rad. Res. 43, 195–205. Trivedi, M.H., Verma, R.J., Sangai, N.P., Chinoy, N.J., 2012. Mitigation by black tea extract of sodium fluoride-induced histopathological changes in brain of mice. Fluoride 45, 13–26. Tunali-Akbay, T., Sehirli, O., Ercan, F., Sener, G., 2010. Resveratrol protects against methorexate-induced hepatic injury in rats. J. Pharm. Pharmaceut. Sci. 13, 303– 310. USNRC, 1993. Health Effects of Ingested Fluoride. National Research Council, National Academy Press, Washington D.C. Vani, M.L., Reddy, K.P., 2000. Effects of fluoride accumulation on some enzymes of brain and gastrocnemius muscle of mice. Fluoride 33, 17–26. Wang, Y., Catana, F., Yang, R., Roderick, R., Van Breemen, R.B., 2002. Analysis of resveratrol in grape products, cranberry juice and wine using liquid chromatography-mass spectrometry. J. Agric. Food Chem. 50, 431–435.
197
Wang, A., Xia, T., Ru, R., Yuan, J., Chen, X., Yang, K., 2004. Antagonistic effect of selenium on oxidative stress, DNA damage, and apoptosis induced by fluoride in human hepatocytes. Fluoride 37, 107–116. Watson, B.D., 1993. Evaluation of the concomitance of lipid peroxidation in experimental models of cerebral ischemia and stroke. Prog. Brain Res. 96, 69– 95. Whitford, G.M., 1983. Fluorides: metabolism, mechanisms action and safety. Dent. Hyg. 57, 16–18. Yan, Y., Yang, J.Y., Mou, Y.H., Wang, L.H., Zhou, Y.N., Wu, C.F., 2012. Differences in the activities of resveratrol and ascorbic acid in protection of ethanol-induced oxidative DNA damage in human peripheral lymphocytes. Food Chem. Toxicol. 50, 168–174. Zhang, W., Xue, J., Ge, M., Yu, M., Liu, L., Zhang, Z., 2013. Resveratrol attenuates hepatotoxicity of rats exposed to arsenic trioxide. Food Chem. Toxicol. 51, 87– 92. Zini, R., Morin, C., Bertelli, A., Bertelli, A.A., Tillement, J.P., 1999. Effects of resveratrol on the rat brain respiratory chain. Drugs Exp. Clin. Res. 25, 87–97.
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Protective effect of resveratrol on sodium fluoride-induced oxidative stress, hepatotoxicity and neurotoxicity in rats Nurgul Atmaca a,⇑, Hasan Tarik Atmaca b, Ayse Kanici c, Tugce Anteplioglu b a
Kirikkale University, Faculty of Veterinary Medicine, Department of Physiology, Kirikkale, Turkey Kirikkale University, Faculty of Veterinary Medicine, Department of Pathology, Kirikkale, Turkey c Kafkas University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Kars, Turkey b
a r t i c l e
i n f o
Article history: Received 7 April 2014 Accepted 15 May 2014 Available online 22 May 2014 Keywords: Fluoride toxicity Oxidative stress Liver Brain Resveratrol Rats
a b s t r a c t Protective effect of resveratrol on sodium fluoride-induced oxidative stress, hepatotoxicity and neurotoxicity were studied in rats. A total of 28 Wistar albino male rats were used. Four study groups were randomly formed with seven animals in each. The groups were treated for 21 days with distilled water (control group), with water containing 100 ppm fluoride (fluoride group), with resveratrol (12.5 mg/kg i.p., resveratrol group), or with 100 ppm fluoride + 12.5 mg/kg resveratrol i.p. (fluoride + resveratrol group). At the end of the trial, blood samples were collected by cardiac puncture and tissue samples were taken simultaneously. The total antioxidant and oxidant status in plasma and tissues as well as plasma 8-hydroxydeoxyguanosine levels were measured. Histopathological analyses of rat liver and brain tissues were performed in all groups to identify any changes. In the fluoride group, the total oxidant levels increased in plasma, liver and brain and total antioxidant levels decreased, as did the plasma 8-hydroxy-deoxyguanosine levels. These changes were prevented by co-administration of resveratrol. In addition, fluoride-associated severe histopathological changes in brain and liver tissues were not observed in the fluoride + resveratrol group. Consequently, these data suggested that resveratrol had beneficial effects in alleviating fluoride-induced oxidative stress. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Fluoride, the most electronegative element, forms ionized fluorides with several other elements (Chlubek, 2003; Rzeuski et al., 1998), and is an important natural and industrial environmental pollutant (Whitford, 1983). Living organisms are exposed to fluoride via food, drinking water, fluoride additives, toothpastes, and professional administration of fluoride gel (Edmunds and Smedley, 1996). Fluorosis is a serious health condition caused by intense and prolonged exposure to inorganic fluoride, which is particularly prevalent in regions where fluoride is released into the air by the burning of fluoride-loaded coal, industrial production of phosphate fertilizers, and volcanic activity (USNRC, 1993), as well
Abbreviations: 8-OHDG, 8-hydroxydeoxyguanosine; MDA, malondialdehyde; NaF, sodium fluoride; TAS, total antioxidant status; TOS, total oxidant status. ⇑ Corresponding author. Address: Kirikkale University, Faculty of Veterinary Medicine, Department of Physiology, 71450, Yahsihan/Kirikkale, Turkey. Tel.: +90 3185374242; fax: +90 318 3573304. E-mail addresses: [email protected] (N. Atmaca), [email protected] (H.T. Atmaca), [email protected] (A. Kanici), [email protected] (T. Anteplioglu). http://dx.doi.org/10.1016/j.fct.2014.05.011 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
as in regions where fluoride-containing groundwater is used for drinking water (Wang et al., 2004). Increased production of reactive oxygen radicals, increased lipid peroxidation, and impaired antioxidant defense mechanisms are involved in the pathogenesis of fluoride toxicity (Vani and Reddy, 2000). Free radicals cause toxicity by attacking membrane phospholipids, leading to membrane injury through lipid peroxidation, depolarization of the mitochondrial membrane, and apoptosis (Barbier et al., 2010). Fluoride crosses the cell membrane (Carlson et al., 1960) and affects soft tissues, including the blood (Karadeniz and Altintas, 2008), brain (Shashi, 2003), and liver (Karaoz et al., 2003; Mittal and Flora, 2006). The liver has an active metabolism that renders it particularly sensitive to fluoride toxicity (Bouaziz et al., 2006; Guo et al., 2007). Many reports demonstrated that fluoride can induce lipid peroxidation and might cause change in the activity of some antioxidant enzymes in liver (Nabavi et al., 2012, 2013; Panneerselvam et al., 2013). Therefore, fluoride-induced oxidative stress and hepatotoxicity are associated with an imbalance in the oxidant/antioxidant systems of liver (Nabavi et al., 2012). Several studies performed on various animal species, including rats, sheep, and cattle, demonstrated that fluoride impaired liver function and
192
N. Atmaca et al. / Food and Chemical Toxicology 70 (2014) 191–197
metabolism, and caused histopathological changes (GruckaMamezar et al., 1997; Kapoor et al., 1993; Kessabi et al., 1986). Also, fluoride can accumulate in the brain (Geeraerts et al., 1986), which leads to abnormal behavioral patterns, impairment of neuronal and cerebrovascular integrity (Mullenix et al., 1995), and metabolic lesions (Vani and Reddy, 2000). Fluoride can also produce detrimental effects in brain tissue by inhibiting enzymes associated with energy production and transport, membrane transport, and synaptic transport (Vani and Reddy, 2000). Previous studies have also shown that fluoride increases lipid peroxidation and alters the levels of antioxidant enzymes in the brain (Flora et al., 2009; Shanthakumari et al., 2004) and liver (Guo et al., 2007; Hassan and Yousef, 2009; Mittal and Flora, 2006). Resveratrol is a compound obtained from the roots of the Polygonum cuspidatum plant used in traditional Eastern medicine for the treatment of fungal diseases, skin inflammation, and cardiovascular and liver diseases (Arichi et al., 1982). Resveratrol (trans3,5,40 -trihydroxystilbene) is a phenolic phytoalexin (Wang et al., 2002) that occurs naturally in various foods, including grapes, plums, cranberries, and peanuts. Its antioxidant effects have been demonstrated in the brain (Ates et al., 2007; Mokni et al., 2007; Sonmez et al., 2007), the liver (Dalaklioglu et al., 2013; TunaliAkbay et al., 2010), and the kidneys (Silan et al., 2007). Resveratrol is a free radical scavenger that increases the activity of several antioxidant enzymes (Gusman et al., 2001; Leonard et al., 2003). Although previous reports have demonstrated that the harmful effects of fluoride reduced because of concomitant intake of antioxidants, including black tea extract (Trivedi et al., 2012), a combination of vitamin E, methionine, and L-carnosine (Agha et al., 2012), Panax ginseng (Karadeniz and Altintas, 2008), pineal proteins, and melatonin (Bharti and Srivastava, 2009), quercetin (Nabavi et al., 2012), gallic acid isolated from Peltiphyllum peltatum (Nabavi et al., 2013), ferulic acid (Panneerselvam et al., 2013). To the best of our knowledge there is no published studies on the protective effects of resveratrol against fluoride toxicity. To address this lack of information, the objective of this study was to investigate the protective effect of resveratrol against oxidative stress caused by fluoride treatment in rats.
2.4. Tissue preparation The livers and brains were removed, washed, and perfused with normal saline to remove residual blood. The liver and brain tissues were homogenized (model TH 220, OMNI, Warrenton, VA, USA) 1:10 (w/v) in ice-cold 140 mM potassium chloride at pH 7.4. The homogenates were centrifuged at 3000 rpm for 10 min at 4 °C, and the supernatants were removed and stored at 80 °C until oxidative stress parameter analyses were performed. 2.5. Total antioxidant status assay Total antioxidant status (TAS) was measured using a commercially available kit from Rel Assay Diagnostics (Gaziantep, Turkey) (Erel, 2004). The method was based on the reduction of colored 2,20 -azino-bis(3-ethylbenzotiazoline-6-sulfonic acid) (ABTS) radical to a colorless reduced form by antioxidants present in the sample. Absorbance was measured spectrophotometrically at a wavelength of 660 nm. The method was calibrated using the vitamin E analog Trolox, and data were expressed as mmol Trolox equivalent (eq.) per liter (mmol Trolox eq./L). 2.6. Total oxidant status assay Total antioxidant status (TOS) was measured using a commercially available kit from Rel Assay Diagnostics (Erel, 2005). The method was based on the principle that the oxidants in the sample oxidized ferrous ions, previously bounded to a chelator, to ferric ions. In the acidic medium of the assay, these ferric ions formed a colored complex with a chromogen. The color intensity was measured spectrophotometrically at a wavelength of 530 nm. This assay was calibrated with hydrogen peroxide (H2O2), and the results were expressed as lmol H2O2 eq./L. 2.7. Determination of plasma 8-hydroxydeoxyguanosine The levels of 8-hydroxydeoxyguanosine (8-OHDG) were determined using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s protocol (Eastbiopharm, Hangzhou, China). The plasma samples were quantified by interpolation using the 8-OHDG standard curve assayed on each plate. The sensitivity limit of the ELISA system was 0.25 ng/mL of 8-OHDG. 2.8. Histopathological analysis Rat liver and brain tissues were fixed in 10% neutral formalin for 48–72 h. The tissues were then trimmed and processed for routine pathological examination. Then, they were embedded in paraffin wax and 4- to 5-lm-thick sections were cut. Hematoxylin and eosin staining was used for all tissue sections. Additionally, a 0.1% cresyl violet staining procedure was used to investigate neurodegenerative changes. Tissue slides were examined under a light microscope (Olympus BX51, Tokyo, Japan).
2. Materials and methods 2.9. Statistical analysis 2.1. Chemicals Sodium fluoride (NaF) was purchased from Merck (Darmstadt, Germany). trans-Resveratrol (>98% purity) was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). All other chemicals were obtained from Merck & Co., Inc. (White House Station, NJ, USA) or Sigma–Aldrich Corporation (St. Louis, MO, USA).
2.2. Animals and treatment Experiments were carried out using male Wistar albino rats weighing 180– 200 g that were fed a standard chow diet with water available ad libitum. Seven animals were housed in each plastic cage under a 12-h light/dark cycle (lights on at 08:00 am) at a constant temperature of 25 ± 2 °C with 42 ± 5% relative humidity. The study protocol was in accordance with the guidelines for animal research and approved by the Ethical Committee of the Kirikkale University (10/155). Twentyeight rats were randomly divided into 4 groups of 7 animals and treated as described below for 21 consecutive days. The control group received distilled water, the fluoride group received drinking water with 100 ppm fluoride, the resveratrol group received daily intraperitoneal (i.p.) administration of 12.5 mg/kg resveratrol, and the fluoride + resveratrol group received drinking water with 100 ppm fluoride and 12.5 mg/kg resveratrol i.p. daily.
2.3. Plasma collection After 21 days, all animals were sacrificed under light ether anesthesia and blood samples were collected into heparinized tubes by cardiac puncture. Plasma was separated by centrifugation at 3000 rpm for 10 min at 4 °C and used for the determination of total antioxidant status (TAS), total oxidant status (TOS), and 8-hydroxydeoxyguanosine (8-OHDG) levels.
Statistical analyses of data were performed using GraphPad Prism 3.0 software (GraphPad Software, San Diego, CA, USA). The data were expressed as mean ± standard error. One-way analysis of variance (ANOVA) was used to analyze the differences between groups. Post hoc comparisons were performed using Tukey’s multiple comparison test. P values less than 0.05 were considered as significant for all statistical calculations.
3. Results 3.1. Oxidative status in the plasma, liver, and brain There were statistically significant differences in TAS, TOS, and 8-OHDG levels between the fluoride group and the control group. Exposure to fluoride significantly elevated plasma TOS and 8-OHDG levels. Additionally, plasma TAS significantly decreased in the fluoride group, compared to those in the other groups. However, in the resveratrol and fluoride + resveratrol groups, these values were not significantly different from those observed in the control group (Table 1). In the fluoride group, TOS significantly increased in liver and brain tissues. In contrast, the TAS of these tissues in the fluoride group was significantly lower than that in the control group. However, TAS and TOS in the resveratrol group and in the fluoride + resveratrol group were not significantly different from those observed in the control group (Table 2). These findings indicated
193
N. Atmaca et al. / Food and Chemical Toxicology 70 (2014) 191–197 Table 1 Oxidative status and 8-OHDG levels in the study groups (n = 7). Parameters TAS (mmol Trolox eq./L) TOS (lmol H2O2 eq./L) 8-OHDG (ng/mL) a,b
Control
Fluoride a
1.14 ± 0.13 4.29 ± 0.65b 0.78 ± 0.09b
Fluoride + resveratrol b
0.76 ± 0.08 7.73 ± 0.38a 1.25 ± 0.08a
ab
0.93 ± 0.09 4.08 ± 1.02b 0.78 ± 0.09b
Resveratrol 1.04 ± 0.04a 3.79 ± 1.21b 0.82 ± 0.06b
The data were expressed as mean ± standard error. Data with a different superscript letter within the same row were significantly different from each other (p < 0.05).
Table 2 Oxidative status in the brain and liver of study groups (n = 7).
a,b
Parameters
Control
Fluoride
Fluoride + resveratrol
Resveratrol
Liver-TAS (mmol Trolox eq./L) Liver-TOS (lmol H2O2 eq./L) Brain-TAS (mmol Trolox eq./L) Brain-TOS (lmol H2O2 eq./L)
2.80 ± 0.23a 7.03 ± 0.48b 0.66 ± 0.03a 4.23 ± 0.14b
2.08 ± 0.14b 8.99 ± 0.38a 0.51 ± 0.01b 5.52 ± 0.22a
2.72 ± 0.10a 7.22 ± 0.33b 0.69 ± 0.03a 4.41 ± 0.29b
2.71 ± 0.09a 7.02 ± 0.48b 0.66 ± 0.04a 4.10 ± 0.26b
The data were expressed as mean ± standard error. Data with a different superscript letter within the same row were significantly different from each other (p < 0.05).
that resveratrol ameliorated the adverse effects of fluoride on TAS and TOS. 3.2. Histopathological assessment of liver and brain Brain histopathological analyses identified no changes in the cerebrum or cerebellum in control and resveratrol groups (Figs. 1 and 2). However, the fluoride group showed significant changes in the histology in various brain regions, and the hippocampus and cerebellum showed neurodegenerative changes. Of all the brain regions examined, the hippocampus showed the most noticeable changes in this study group. Neurons were shrunken and darkly stained, with small nuclei, and cell number decreased.
The Purkinje cells in the cerebellum were the most affected cell population in the fluoride group (Fig. 2c). The cerebrum and cerebellum of the fluoride + resveratrol group (Figs. 1d and 2d) showed structures similar to those in the control group. Histopathological examination of the liver showed normal structures in the control and resveratrol groups (Fig. 3a and b). However, the fluoride group showed pathological liver changes, including hydropic and vacuolar degeneration, and some necrosis. Slight liver congestion was also observed, as manifested by central vein and mild sinusoidal distension and occasional central hepatocyte degeneration (Fig. 3c). The fluoride + resveratrol group had liver tissue histology similar to that in the control group (Fig. 3d).
Fig. 1. Histological sections of the hippocampus of the study groups. (a) Control group hippocampus stained with hematoxylin and eosin (main panels) or cresyl violet (inset panels). Arrows indicate normal neurons and arrowheads indicate cresyl violet-stained neurons. The magnification was 200. (b) Resveratrol group hippocampus stained with hematoxylin and eosin (main panels) or cresyl violet (inset panels). Arrows indicate normal neurons and arrowheads indicate cresyl violet-stained neurons. The magnification was 200. (c) Fluoride group hippocampus stained with hematoxylin and eosin (main panels) or cresyl violet (inset panels). Note the degenerated neurons (arrows) and dark cresyl violet-stained shrunken neurons. The magnification was 200. (d) Fluoride + resveratrol group hippocampus stained with hematoxylin and eosin (main panels) or cresyl violet (inset panels). Note that the neurons appear normal and the number of degenerated neurons number was marginally less than that in the fluoride group (arrow). Some darkly stained shrunken neurons were observed using cresyl violet staining (arrow heads). The magnification was 200. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
194
N. Atmaca et al. / Food and Chemical Toxicology 70 (2014) 191–197
Fig. 2. Histological sections of the cerebellum of the study groups. (a) Control group cerebellum stained with cresyl violet. Normal neurons are indicated by arrows. The magnification was 200. (b) Resveratrol group cerebellum stained with cresyl violet. Normal neurons are indicated by arrows. The magnification was 200. (c) Flouride group cerebellum stained with cresyl violet. Darkly stained shrunken neurons are indicated by arrows. The magnification was 200. (d) Fluoride + resveratrol group cerebellum stained with cresyl violet. Note that the neurons had a normal appearance, and the number of degenerated (shrunken) neurons was marginally less than that observed in the fluoride group (arrows). The magnification was 200. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Histological sections of the liver of the study groups. (a) Control group liver stained with hematoxylin and eosin. The liver structure appeared normal. Vc = central vein. The magnification was 40. (b) Resveratrol group liver stained with hematoxylin and eosin. Liver structure appeared normal. Vc = central vein. The magnification was 40. (c) Fluoride group liver stained with hematoxylin and eosin. The asterisk indicates necrotic tissue, arrows indicate vacuolar degeneration, and hyperemia can be seen in sinusoids (arrow heads). Vc = central vein. The magnification was 200. (d) Fluoride + resveratrol group liver stained with hematoxylin and eosin. Some vacuolar degeneration was observed (arrow); however, in general, the tissue structure appeared normal. Vc = central vein. The magnification was 200.
4. Discussion 4.1. The resveratrol group Compared to the control group, the resveratrol group showed no statistically significant changes in oxidative stress parameters,
including plasma, liver, and brain TAS and TOS, and plasma 8-OHDG level. A previous study of the dose-dependent effects of resveratrol in the brain tissue of healthy rats reported decreased levels of malondialdehyde (MDA) in the brain, and increased antioxidant enzymes activities such as superoxide dismutase, catalase, and peroxidase (Mokni et al., 2007). The dose of resveratrol used in
N. Atmaca et al. / Food and Chemical Toxicology 70 (2014) 191–197
the present study was selected in line with this report, where the optimal dose of resveratrol for effects on antioxidant enzymes was 12.5 mg/kg (Mokni et al., 2007). The lack of any adverse oxidative stress-related effects of resveratrol in the present study was likely due to the strong antioxidant (Ignatowicz and BearDubowska, 2001), hepatoprotective (Kasdallah-Grissa et al., 2007; Tunali-Akbay et al., 2010), and neuroprotective (Bastianetto et al., 2000) effects of this substance. 4.2. The fluoride group Increased free radical production plays a major role in the pathogenesis of several diseases and toxicities (Halliwell, 1992). Studies have suggested that fluoride increases free radical production, and that these free radicals mediate its toxic effects on soft tissues (Rzeuski et al., 1998). In this study, the fluoride group showed increased TOS in plasma, liver, and brain tissue. This increase was statistically significant when compared to the control group. Furthermore, the fluoride group had significantly decreased TAS in plasma, and liver and brain tissues. These alterations indicated that a high dose of fluoride (100 ppm) administered for a 21-day period induced free radical formation that was not neutralized by endogenous antioxidant systems. The observed increase in TOS is also supportive of this finding. Reduced TAS might reflect the consumption of endogenous antioxidant molecules or a fluorideinduced inhibition of antioxidant enzyme activity. Fluoride has been shown to induce lipid peroxidation and decrease levels of several antioxidant molecules and enzyme activities in the blood (Hassan and Abdel-Aziz, 2010), the liver (Hassan and Yousef, 2009; Hassan and Abdel-Aziz, 2010; Nabavi et al., 2012, 2013; Panneerselvam et al., 2013), and the brain (Hassan and Abdel-Aziz, 2010). However, some studies have reported that fluoride did not affect antioxidant defense systems (Reddy et al., 2003). The current study and others have demonstrated that fluoride decreased total plasma antioxidant capacity in treated animals (Bouaziz et al., 2006; Hassan and Yousef, 2009; Hassan and AbdelAziz, 2010; Inkielewicz-Stepniak and Czarnowski, 2010). Fluoride has also been shown to produce marked increases in MDA and reactive products of thiobarbituric acid, which are indicators of lipid peroxidation caused by increased free radicals (Eraslan et al., 2007; Hassan and Yousef, 2009; Hassan and Abdel-Aziz, 2010; Inkielewicz-Stepniak and Czarnowski, 2010). In the present study, the increase in TOS, which indicated the total amount of reactive oxygen metabolites in plasma, and liver and brain tissues, and the decrease in TAS, suggested that the balance between oxidative and antioxidant systems was impaired in rats exposed to fluoride (Hassan and Abdel-Aziz, 2010), which resulted in oxidative stress. Additionally, fluoride administration increased levels of 8-OHDG, a marker of oxidative DNA injury (Loft and Poulsen, 1996). 8-OHDG is the main product of a hydroxyl radical attack on guanosine monophosphates (Harman, 1992), and is primarily removed from cells via DNA repair mechanisms (Burkhardt et al., 2001). The current study suggested that DNA damage occurred in fluoride-treated rats that DNA repair mechanisms were unable to counteract. In addition, histological analyses of the liver and the brain indicated that fluoride produced toxic effects on soft tissues. In the fluoride group, necrotic areas and hyperemia of the sinusoids were observed in liver tissue, as were degenerative neurons in the hippocampus and neural shrinkage in the cerebellum. The liver and the brain are particularly sensitive to free radical damage; liver tissue is highly metabolically active (Shashi and Thaper, 2000), and the multiple unsaturated fatty acid content in the brain produces an increased oxygen requirement (Watson, 1993). Fluoride has previously been reported to cause degenerative changes, ranging from balloon degeneration to hepatocellular necrosis and mononu-
195
clear cell infiltration, in the liver tissue of mice (Bouaziz et al., 2006) and rats (Karaoz et al., 2003; Panneerselvam et al., 2013). In addition, the brain has a low iron-binding capacity, as well as a significant level of iron ions that facilitate free radical production (Halliwell, 1992). Consistent with the findings of the present study, it has been reported that fluoride administration in rabbits caused chromatolysis and nuclear splitting in Purkinje cells, and shrunken, picnotic, and hyperchromatic neurons (Shashi, 2003). Fluoride administration in mice caused neurodegenerative changes in the cerebral hemisphere, cerebellum, and medulla oblongata, as well as chromatolysis, picnotic nuclei, fatty infiltration, and vacuolization (Trivedi et al., 2012). The neurodegenerative changes observed in the fluoride group suggested that fluoride crossed the blood– brain barrier and damaged neurons via oxidative changes. 4.3. The fluoride + resveratrol group Resveratrol is both a free radical scavenger and a potent antioxidant because of its ability to promote the activities of a variety of antioxidant enzymes (Alarcón de la Lastra et al., 2006). In addition, some investigators have indicated a potential hepatoprotective (Kasdallah-Grissa et al., 2007; Tunali-Akbay et al., 2010; Zhang et al., 2013) and neuroprotective (Ates et al., 2007; Sonmez et al., 2007; Lu et al., 2008) activities for resveratrol based on its beneficial effects in several liver and brain damage models. In this study, plasma TOS and TAS did not differ significantly between the fluoride + resveratrol group and the control group. However, plasma TOS in the fluoride + resveratrol group significantly decreased in comparison with that in the fluoride group, and TAS tended to increase, although not significantly. In addition, liver and brain tissue TOS and TAS were similar to those of the control group, but in both tissues, TOS was lower and TAS was higher, than those in the fluoride group. These plasma and liver, and brain tissue observations demonstrated the free radical scavenger and antioxidant effects of resveratrol. The effects of resveratrol on these parameters may be associated with its constituent compounds. Resveratrol is a polyphenolic compound and the most active of the stilbene phytoalexins (Kolouchova-Hanzlikova et al., 2004). The ability of the polyphenolic compounds to act as antioxidants depends on the redox properties of their phenolic hydroxyl groups and the potential for electron delocalization across the chemical structure (Ignatowicz and BearDubowska, 2001). Furthermore, the high hydrophilic and lipophilic content of resveratrol plays an important role in its efficacy in comparison to other antioxidants such as vitamins C and E (Kasdallah-Grissa et al., 2007). In this study, the antioxidant effects of resveratrol were considered to result from 3 mechanisms: first, the reduction of oxidative chain reactions of free oxygen species in competition with coenzyme Q; second, neutralization of superoxide radicals formed in the mitochondria; and third, prevention of lipid peroxidation by Fenton reaction products (Zini et al., 1999). The decrease observed in 8-OHDG levels, a biomarker of oxidative DNA injury, in the fluoride + resveratrol group compared to the fluoride group (Loft and Poulsen, 1996), also provided evidence for the antioxidant and free radical-scavenging activities of resveratrol. Similar to this study, resveratrol has been reported to play an effective role in preventing oxidative DNA injury in vivo (Guo et al., 2007; Tatlidede et al., 2009) and in vitro (Yan et al., 2012). Furthermore, the present study found that liver and brain tissues were generally normal in structure in the fluoride + resveratrol group, in contrast to the abnormal changes seen in the fluoride group. These results were consistent with reports of the strong antioxidant (Gulcin, 2010), hepatoprotective (Kasdallah-Grissa et al., 2007; Tunali-Akbay et al., 2010; Zhang et al., 2013), and neuroprotective (Ates et al., 2007; Mokni et al., 2007; Sonmez et al., 2007) activities of resveratrol.
196
N. Atmaca et al. / Food and Chemical Toxicology 70 (2014) 191–197
5. Conclusions These results have shown that oxidative stress participated in fluoride-induced toxicity, and resveratrol protected against fluoride-induced oxidative stress and tissue injury. Therefore, these results suggest that resveratrol inhibits the development of fluoride-induced hepatotoxicity and neurotoxicity in rats. Future studies will explore this effect in other model organisms, establish its dose–response relationship, and further examine the mechanisms involved. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version. Acknowledgment Financial support for this Project (Project No: 2011/44) was provided by the Kirikkale University Research Fund. References Agha, F.E., El-Badry, M.O., Hassan, D.A.A., Elraouf, A.A., 2012. Role of vitamin E combination with methionine and L-carnosine against sodium fluoride-induced hematological, biochemical, DNA damage, histological and immunohistochemical changes in pancreas of albino rats. Life Sci. J. 9, 1260–1275. Alarcón de la Lastra, C., Villegas, I., Martín, A.R., 2006. In: Aggarwald, B.B., Shishodia, S. (Eds.), Resveratrol in Health and Disease. CRC Press, Boca Raton, pp. 33–56. Arichi, H., Kimura, Y., Okuda, H., Baba, K., Kozawa, M., Arichi, S., 1982. Effects of stilbene components of the roots of Polygonum cuspidatum Sieb et Zucc on lipid metabolism. Chem. Pharm. Bull. 30, pp. 1776–1770. Ates, O., Cayli, S., Altinoz, E., Gurses, I., Yucel, N., Sener, M., Kocak, A., Yologlu, S., 2007. Neuroprotection by resveratrol against traumatic brain injury in rats. Mol. Cell. Biochem. 294, 137–144. Barbier, O., Arreola-Mendoza, L., Del Razo, L.M., 2010. Molecular mechanisms of fluoride toxicity. Chem-Biol. Interact. 188, 319–333. Bastianetto, S., Zheng, W.H., Quirion, R., 2000. Neuroprotective abilities of resveratrol and other red wine constituents against nitric oxide related toxicity in cultured hippocampal neurons. Br. J. Pharmacol. 131, 711–720. Bharti, V.K., Srivastava, R.S., 2009. Fluoride-induced oxidative stress in rat’s brain and its amelioration by Buffalo (Bubalus bubalis) pineal proteins and melatonin. Biol. Trace Elem. Res. 130, 131–140. Bouaziz, H., Ketata, S., Jammoussi, K., Boudawara, T., Ayedi, F., Ellouze, F., Zeghal, N., 2006. Effects of sodium fluoride on hepatic toxicity in adult mice and their suckling pups. Pestic. Biochem. Phys. 86, 124–130. Burkhardt, S., Reiter, R.J., Tan, D., 2001. DNA oxidatively damaged by chromium and H2O2 is protected by the antioxidants melatonin, N1-acetyl-N2-formayl-5methoxykynura mine, resveratrol and uric acid. Int. J. Biochem. Cell. 33, 775– 783. Carlson, C.H., Armstrong, W.D., Singer, L., 1960. Distribution and excretion of radio fluoride in the human. Proc. Soc. Exp. Biol. Med. 104, 235–239. Chlubek, D., 2003. Fluoride and oxidative stress. Fluoride 36, 217–228. Dalaklioglu, S., Genc, G.E., Aksoy, N.H., Akcit, F., Gumuslu, S., 2013. Resveratrol ameliorates methotrexate-induced hepatotoxicity in rats via inhibition of lipid peroxidation. Hum. Exp. Toxicol. 32, 662–667. Edmunds, W.M., Smedley, P.L., 1996. Groundwater geochemistry and health: an overview. Environ. Geochem. Health 113, 91–105. Eraslan, G., Kanbur, M., Silici, S., 2007. Evaluation of propolis effects on some biochemical parameters in rats treated with sodium fluoride. Pestic. Biochem. Phys. 88, 273–283. Erel, O., 2004. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin. Biochem. 37, 277–285. Erel, O., 2005. A new automated colorimetric method for measuring total oxidant status. Clin. Biochem. 38, 1103–1111. Flora, S.J.S., Mittal, M., Mishra, D., 2009. Co-exposure to arsenic and fluoride on oxidative stress, glutathione linked enzymes, biogenic amines and DNA damage in Mouse brain. J. Neurol. Sci. 285, 198–205. Geeraerts, F., Gijs, G., Finne, E., Crokaert, R., 1986. Kinetics of fluoride penetration in liver and brain. Fluoride 19, 108–112.
Grucka-Mamezar, E., Machoy, A., Tarnawski, R., Birkner, E., Mamezar, A., 1997. Influence of long term sodium fluoride administration on selected parameters of rat blood serum and liver function. Fluoride 30, 157–164. Gulcin, I., 2010. Antioxidant properties of resveratrol: a structure-activity insight. Innov. Food Sci. Emerg. 11, 210–218. Guo, L., Wang, L.H., Sun, B.S., Zhao, Y.Q., Dong, Y.X., Wu, C.F., 2007. Direct in vivo evidence of protective effects of grape seed procyanidin fractions and other antioxidants against ethanol-induced oxidative DNA damage in Mouse brain cells. Agric. Food Chem. 55, 5881–5891. Gusman, J., Malonne, H., Atassi, G., 2001. A reappraisal of the potential chemopreventive and chemotherapeutic properties of resveratrol. Carcinogenesis 22, 1111–1117. Halliwell, B., 1992. Reactive oxygen species and the central nervous system. J. Neurochem. 59, 1609–1623. Harman, D., 1992. Free radical theory of aging. Mutat. Res. 275, 257–266. Hassan, H.A., Abdel-Aziz, A.F., 2010. Evaluation of free radical-scavenging and antioxidant properties of black berry against fluoride toxicity in rats. Food Chem. Toxicol. 48, 1999–2004. Hassan, H.A., Yousef, M., 2009. Mitigating effects of antioxidant properties of black berry juice on sodium fluoride induced hepatotoxicity and oxidative stress in rats. Food Chem. Toxicol. 47, 2332–2337. Ignatowicz, E., Bear-Dubowska, W., 2001. Resveratrol, a natural chemopreventive agent against degenerative diseases. Pol. J. Pharmacol. 53, 557–569. Inkielewicz-Stepniak, I., Czarnowski, W., 2010. Oxidative stress parameters in rats exposed to fluoride and caffeine. Food Chem. Toxicol. 48, 1607–1611. Kapoor, V., Prasad, T., Bhatia, K.C., 1993. Effect of dietary fluorine on histopathological changes in calves. Fluoride 26, 105–110. Karadeniz, A., Altıntas, L., 2008. Effects of Panax Ginseng on fluoride-induced haematological pattern changes in mice. Fluoride 41, 67–71. Karaoz, E., Gulle, K., Mumcu, E.F., Gokcimen, A., Oncu, M., 2003. Deneysel kronik florozis olusßturulmusß 2. Kusßak sıçan böbrek ve karacig˘er dokularında yapısal deg˘isßiklikler. Turk. Klin. Tıp Bilim. 23, 129–134. Kasdallah-Grissa, A., Mornagui, B., Aouani, E., Hammami, M., El May, M., Gharbi, N., Kamoun, A., El-Fazaa, S., 2007. Resveratrol, a red wine polyphenol, attenuates ethanol-induced oxidative stress in rat liver. Life Sci. 80, 1033–1039. Kessabi, M., Hamliri, A., Braun, J.P., 1986. Experimental fluorosis in sheep: alleviating effects of aluminium. Vet. Hum. Toxicol. 28, 300–304. Kolouchova-Hanzlikova, I., Melzoch, K., Filip, V., Smidrkal, J., 2004. Rapid method for resveratrol determination by HPLC with electrochemical and UV detections in wines. Food Chem. 87, 151–158. Leonard, S., Xia, C., Jiang, B.H., Stinefelt, B., Klandorf, H., Harris, G.K., Shi, X., 2003. Resveratrol scavenges reactive oxygen species and effects radical-induced cellular responses. Biochem. Biophys. Res. Commun. 309, 1017–1026. Loft, S., Poulsen, H.E., 1996. Cancer risk and oxidative DNA damage in man. J. Mol. Med. 74, 297–312. Lu, K.T., Ko, M.C., Chen, B.Y., Huang, J.C., Hsieh, C.W., Lee, M.C., Chiou, R.Y.Y., Wung, B.S., Peng, C.H., Yang, Y.L., 2008. Neuroprotective effects of resveratrol on MPTPinduced neuron loss mediated by free radical scavenging. J. Agric. Food Chem. 56, 6910–6913. Mittal, M., Flora, S.J.S., 2006. Effects of individual and combined exposure to sodium arsenite and sodium fluoride on tissue oxidative stress, arsenic and fluoride levels in male mice. Chem-Biol. Interact. 162, 128–139. Mokni, M., Elkahoui, S., Limam, F., Amri, M., Aouani, E., 2007. Effect of resveratrol on antioxidant enzyme activities in the brain of healthy rat. Neurochem. Res. 32, 981–987. Mullenix, P.J., Enbesten, P.K., Schunior, A., Kernan, W.J., 1995. Neurotoxicity of sodium fluoride in rats. Neurotoxicol. Teratol. 17, 169–177. Nabavi, S.M., Nabavi, S.F., Eslami, S., Moghaddam, A.H., 2012. In vivo protective effects of quercetin against sodium fluoride-induced oxidative stress in the hepatic tissue. Food Chem. 132, 931–935. Nabavi, S.F., Nabavi, S.M., Habtemariam, S., Moghaddam, A.H., Sureda, A., Jafari, M., Latifi, A.M., 2013. Hepatoprotective effect of gallic acid isolated from Peltiphyllum peltatum against sodium fluoride-induced oxidative stress. Ind. Crops Prod. 44, 50–55. Panneerselvam, L., Subbiah, K., Arumugam, A., Senapathy, J.G., 2013. Ferulic acid modulates fluoride-induced oxidative hepatotoxicity in male Wistar rats. Biol. Trace Elem. Res. 151, 85–91. Reddy, G.B., Khandare, A.L., Reddy, P., Rao, G.S., Balakrishna, N., Srivalli, I., 2003. Antioxidant defence system and lipid peroxidation in patients with skeletal fluorosis and fluoride-intoxicated rabbits. Toxicol. Sci. 72, 363–368. Rzeuski, R., Chlubek, D., Machoy, Z., 1998. Interactions between fluoride and biological free radical reactions. Fluoride 31, 43–45. Shanthakumari, D., Sirinivasalu, S., Subramanian, S., 2004. Effect of fluoride intoxication on lipid peroxidation and antioxidant status in experimental rats. Toxicology 204, 219–228. Shashi, A., 2003. Histopathological investigation of fluoride-induced neurotoxicity in rabbits. Fluoride 36, 95–105. Shashi, A., Thaper, S.P., 2000. Histopathology of fluoride-induced hepatotoxicity in rabbits. Fluoride. 34, 34–42. Silan, C., Uzun, O., Comunog˘lu, N.U., Golcen, S., Bedirhan, S., Cengiz, M., 2007. Gentamicin-induced nephrotoxicity in rats ameliorated and healing effects of resveratrol. Biol. Pharm. Bull. 30, 79–83. Sonmez, U., Sonmez, A., Erbil, G., Tekmen, I., Baykara, B., 2007. Neuroprotective effects of resveratrol against traumatic brain injury in immature rats. Neurosci. Lett. 420, 133–137.
N. Atmaca et al. / Food and Chemical Toxicology 70 (2014) 191–197 Tatlidede, E., Sehirli, O., Velioglu-Ogunç, A., Cetinel, S., Yegen, B.C., Yarat, A., Süleymanoglu, S., Sener, G., 2009. Resveratrol treatment protects against doxorubucin-induced cardiotoxicity by alleviating oxidative damage. Free Rad. Res. 43, 195–205. Trivedi, M.H., Verma, R.J., Sangai, N.P., Chinoy, N.J., 2012. Mitigation by black tea extract of sodium fluoride-induced histopathological changes in brain of mice. Fluoride 45, 13–26. Tunali-Akbay, T., Sehirli, O., Ercan, F., Sener, G., 2010. Resveratrol protects against methorexate-induced hepatic injury in rats. J. Pharm. Pharmaceut. Sci. 13, 303– 310. USNRC, 1993. Health Effects of Ingested Fluoride. National Research Council, National Academy Press, Washington D.C. Vani, M.L., Reddy, K.P., 2000. Effects of fluoride accumulation on some enzymes of brain and gastrocnemius muscle of mice. Fluoride 33, 17–26. Wang, Y., Catana, F., Yang, R., Roderick, R., Van Breemen, R.B., 2002. Analysis of resveratrol in grape products, cranberry juice and wine using liquid chromatography-mass spectrometry. J. Agric. Food Chem. 50, 431–435.
197
Wang, A., Xia, T., Ru, R., Yuan, J., Chen, X., Yang, K., 2004. Antagonistic effect of selenium on oxidative stress, DNA damage, and apoptosis induced by fluoride in human hepatocytes. Fluoride 37, 107–116. Watson, B.D., 1993. Evaluation of the concomitance of lipid peroxidation in experimental models of cerebral ischemia and stroke. Prog. Brain Res. 96, 69– 95. Whitford, G.M., 1983. Fluorides: metabolism, mechanisms action and safety. Dent. Hyg. 57, 16–18. Yan, Y., Yang, J.Y., Mou, Y.H., Wang, L.H., Zhou, Y.N., Wu, C.F., 2012. Differences in the activities of resveratrol and ascorbic acid in protection of ethanol-induced oxidative DNA damage in human peripheral lymphocytes. Food Chem. Toxicol. 50, 168–174. Zhang, W., Xue, J., Ge, M., Yu, M., Liu, L., Zhang, Z., 2013. Resveratrol attenuates hepatotoxicity of rats exposed to arsenic trioxide. Food Chem. Toxicol. 51, 87– 92. Zini, R., Morin, C., Bertelli, A., Bertelli, A.A., Tillement, J.P., 1999. Effects of resveratrol on the rat brain respiratory chain. Drugs Exp. Clin. Res. 25, 87–97.