Introduction

Fluoride is one of the widespread environmental pollutants, especially in industrial areas (Sun et al., 2017). It becomes a known reason for severe water contamination due to its highly electronegative nature and prevalence (Sharma et al., 2017). Ingesting fluoride from drinking water is the most common way of fluoride toxicity. Hence, WHO suggests fluoride level should be below 1.5 mg/L in drinking water to prevent various disorders (Sun et al., 2016). Its harmful or beneficial effects depend on the concentration of fluoride in the body (Sun et al., 2017). Exorbitant fluoride ingestion, mostly through the drinking water, may have detrimental impacts on teeth, bone, heart, kidney, liver and brain (Ma, Zhang, Ren, & Wang, 2017; Sun et al., 2017). Available data suggest that excessive fluoride exposure leads to its accumulation in different parts of the brain and gastrocnemius muscle and contribute to disturbed oxygen metabolism and ROS formation, thereby induces oxidative stress, which in turn, alters the phospholipids metabolism leading to muscular and neuronal damage and death (Mesram, Nagapuri, Banala, Nalagoni, & Karnati, 2017; Nalagoni & Karnati, 2016). In addition to stimulating oxidative stress, fluoride also reduces the antioxidant potential of various organs by decreasing the activity of antioxidant enzymes (Bhatnagar, Sharma, Suhalka, Sukhwal, & Jaiswal, 2014).

Dietary supplements trap the reactive oxygen species (Jiang, 2014; Ju et al., 2010) and thereby causing an increase in antioxidant vitamin levels (Batista et al., 2016). Therefore, the importance of dietary supplementation in boosting up the antioxidative defence has been attracted for decades. Selenium (Se) is a non-metallic trace element known as a nutrient essential to human health (Klein et al., 2003). It regulates the synthesis of selenoproteins. Selenoproteins involve in antioxidant defences by acting as a cofactor for the antioxidant enzymes to prevent oxidative injury (Cardoso, Bandeira, Jacob-Filho, & Cozzolino, 2014; Nazıroglu, 2009).

Further, it has been suggested as a neuroprotectant in animal models of Alzheimer’s disease (Lakshmi, Sudhakar, & Prakash, 2015). Alpha-tocopherol acts as a major lipid-soluble antioxidant in cell membranes. It specifically inhibits lipid peroxidation by scavenging various types of free radicals (Uneri, Sari, Akboga, & Yuksel, 2006). Several studies reported a decrease in oxidative stress by individual and combined antioxidant supplementation (Block, Jensen, Morrow, Holland, & Norkus, 2008; Devaraj, Leonard, Traber, & Jialal, 2008). Supplementation with selenium, vitamin C, and vitamin E effectively improved antioxidant–oxidant balance in obese children and adolescents (Murer et al., 2014). It is known that in combination, Se and α-tocopherol act synergistically against the formation of reactive oxygen species (Pak, Lanteri, Scheuch, & Sawczuk, 2002). The above information prompted us to examine the antioxidant capacity of Se and alpha-tocopherol. Thus, in this study, we intended to explore the protective effects of selenium and alpha-tocopherol individually and combinedly on oxidative stress in the brain and gastrocnemius muscle of experimental animals.

Materials and Methods

Chemicals

Selenium and sodium fluoride from Loba Chemie and alpha-tocopherol from Merck Company were purchased.

Animal maintenance

Swiss albino mice (30±2g) were purchased from NIFLA, NIN, Hyderabad, India. The animals were housed under standard conditions (22±2^oC, 12 hrs light-dark cycle, pellet diet and water).

Experimental design

The animals were divided into five groups (6 animals each). Group-1: Saline, Group-2: NaF (20mg/ kg BW), Group-3: Selenium (5 µg/ kg BW), Group-4: Alpha-tocopherol (2 mg/kg BW) Group-5: NaF+Se+alpha-tocopherol (20 mg/kg BW+5 µg/kg BW+2mg/kg BW). The doses were given daily between 8.15 am–9.15 am for fifteen days. All the experiments were conducted as per the institutional ethical committee guidelines (No: 383/01/a/CPCSEA).

Biochemical assays

The isolated tissues were homogenised, and the supernatant was collected for biochemical studies. XOD activity was measured as described by (Govindappa & Swami, 1966). The results were expressed as µ moles of formazan formed/mg protein/hr. Lipid peroxide formation was measured by using Wills method (Wills, 1966). MDA is the end product of peroxidised polyunsaturated fatty acids. The result was expressed as nmol of MDA/mg protein/3mins. SOD activity was analysed as described by the (Kono, 1978). The results were expressed in units (U/mg protein). Catalase activity was assayed as described by (Luck, 1971). The result was expressed as μMol of H₂O₂ decomposed/min/mg/protein. The GST activity was assayed as described by (Habig, Pabst, & Jakoby, 1974). The results were expressed as n mole of GS-CDNB/mg protein/min. Glutathione peroxidase activity was measured as described by (Lawrence & Burk, 1976). The result was expressed as μ moles of NADPH oxidised/min/mg/protein.

Statistical analysis

The data were analysed using one-way ANOVA, followed by Tukey's studentised range test (HSD). The significant level was considered at p=0.05.

Results and Discussion

Xanthine oxidase activity

The XOD activity was significantly (p<0.05) enhanced in the brain (27.95%) and muscle (27.98%) in the fluoride group in comparison with the control group. In contrast, it was recovered partially in NaF+selenium, NaF+α-tocopherol and significantly (p<0.05) in NaF+Selenium+ α-tocopherol treated groups with respect of 24.73%, 25.80% and 1.61% in the brain and 26.10%, 24.84% and 1.88% in muscle indicates synergistic effects of Se and α-tocopherol (Figure 1).

Lipid peroxidation

The MDA content was significantly (p<0.05) enhanced in the brain (25.96%) and muscle (30.36%) in the fluoride group in comparison with the control group. In contrast, it was recovered partially in NaF+selenium, NaF+ α-tocopherol and significantly (p<0.05) in NaF+Selenium+α-tocopherol treated groups with respect of 22.69%, 22.09% and 3.32% in brain and 27.17%, 26.70% and 3.45% in muscle indicates synergistic effects of Se and α-tocopherol (Figure 2).

Superoxide dismutase (SOD)

The SOD activity was significantly (p<0.05) decreased in the brain (23.21%) and muscle (32.39%) in the fluoride group in comparison with the control group. In contrast, it was recovered partially in NaF+Selenium, NaF+ α-tocopherol and significantly (p<0.05) in NaF+Selenium+α-tocopherol treated groups with respect of 19.46%, 20.89% and 2.14% in brain and 29.57%, 30.78% and 1.40% in muscle indicates synergistic effects of Se and α-tocopherol (Figure 3).

Catalase (CAT)

The catalase activity was significantly (p<0.05) decreased in the brain (21.61%) and muscle (21.68%) in the fluoride group in comparison with the control group. In contrast, it was recovered partially in NaF+selenium, NaF+ α-tocopherol and significantly (p<0.05) in NaF+Selenium+α-tocopherol treated groups with respect of 15.67%, 18.22% and 2.11% in brain and 15.48%, 19.02% and 2.21% in muscle indicates synergistic effects of Se and α-tocopherol (Figure 4).

Glutathione-S-Transferase (GST)

The GST activity was significantly (p<0.05) decreased in the brain (8.93%) and muscle (13.77%) in the fluoride group in comparison with the control group. In contrast, it was recovered partially in NaF+Selenium, NaF+ α-tocopherol and significantly (p<0.05) in NaF+Selenium+α-tocopherol treated groups with respect of 3.83%, 5.42% and 1.59% in brain and 10.22%, 12.59% and 4.72% in muscle indicates synergistic effects of Se and α-tocopherol (Figure 5).

Glutathione peroxidase (GPx)

The GPx activity was significantly (p<0.05) declined in the brain (26.82%) and muscle (30.73%) in the fluoride group in comparison with the control group. In contrast, it was recovered partially in NaF+selenium, NaF+ α-tocopherol and significantly (p<0.05) in NaF+Selenium+α-tocopherol treated groups with respect of 18.85%, 21.85% and 2.91% in brain and 24.63%, 25.15% and 3.36% in muscle indicates synergistic effects of Se and α-tocopherol (Figure 6).

Fluoride is an irresistible environmental toxic element. Its excessive exposure induces several adverse effects on human health (Meenakshi & Maheshwari, 2006). Ample of studies on animals and humans chronically exposed to fluoride reported vulnerability of all soft tissue organs (Bhatnagar, Rao, & Saxena, 2006). Fluoride accumulation in the brain and muscle of mice induces stress and prevents oxidative defence mechanisms, thereby resulting in oxidative stress-induced neuronal and muscular tissues damage (Vani & Reddy, 2000). Fluoride, a known neurotoxic agent, affects brain function and development (Ge et al., 2018; McPherson et al., 2018). Repeated exposure to fluoride induces pathological lesions in the brain of experimental animals (Adedara et al., 2017; Khan, Raina, Dubey, & Verma, 2018). This study is reporting the antioxidant effects of Se and α-tocopherol on oxidative stress in the brain and muscle of experimental mice models.

It is well known that fluoride toxicity induces ROS production (Apel & Hirt, 2004). ROS includes hydrogen peroxide, superoxide and hydroxyl radicals etc. These free radicals are mainly generated by redox reactions catalysed by xanthine oxidase (XO), flavin oxidase, NADPH oxidase (NOX), cytochrome P450, and also by respiratory chain components in mitochondria (Dasuri, Zhang, & Keller, 2013). In the present study, the increased xanthine oxidase activity (p<0.05) in the brain and muscle of fluoride exposure group in comparison with the control group confirms the increased ROS production by fluoride toxicity. The increased ROS production leads to lipid peroxidation, which is indicated by increased malondialdehyde content (Farmer & Mueller, 2013; Oyagbemi et al., 2020). In the current study, the increased MDA content (p<0.05) in the brain and muscle of NaF group in comparison with the control group indicates the lipid peroxidation induced by increased reactive oxygen species. The increased ROS level reduces the activity of the antioxidant enzymes. Antioxidant enzymes include SOD, CAT, GST, GPx, GSH, etc., play a key role in the elimination of ROS, thereby decreasing oxidative stress. SOD and CAT are blockers of free radicals generated by toxic substances. SOD plays a crucial role in the first line of defence. It dismutases the peroxides into H₂0₂ and O₂. Catalase removes H₂O₂ by converting it into O₂ and H₂O. In the present study, SOD and catalase activities were significantly (p<0.05) decreased in muscle and brain in NaF treated group in comparison with the control group. The results are following earlier studies which reported declined SOD and CAT activities in the gastrocnemius muscle, liver, heart, kidney and brain in fluoridated animals (Nalagoni et al., 2016; Oyagbemi et al., 2020) and people living in endemic fluoride areas (Li & Cao, 1994). GST and GPx activities were significantly (p<0.05) decreased in brain and muscle in fluoride intoxicated group in comparison with the control group. The decline in their activities could be a result of their augmented utilisation to purge hydrogen peroxide and organic hydroperoxides from both the tissues. The present study results conform with the previous studies where fluoride inhibits the activity of the antioxidant enzymes includes SOD, CAT, GPx, GST and GR (Oyagbemi et al., 2020; Sarkar, Pal, Das, & Dinda, 2014). However, the Se and Alpha-tocopherol combination significantly (p<0.05) declined the elevated MDA Content, inhibited xanthine oxidase activity and restored the activities of antioxidant enzymes such as SOD, CAT, GPx, and GST than individual administration. The protective effects of Se and alpha-tocopherol could be due to the trapping free radicals and restoring the antioxidant defence (Jiang, 2014; Meydani, John, Macauley, & Jeffrey, 1988). Collectively, the results indicating that the muscle was more prone to the fluoride toxicity than the brain, which may be due to the protective role of the blood-brain barrier and differential sensitivity of various organs to the fluoride toxicity. However, Se and alpha-tocopherol synergistically mitigated the oxidative stress in the brain and muscle in fluoridated animals.

Conclusions

In conclusion, the fluoride exposure resulted in a significant increase in XOD activity, enhanced LPO and a decline in the antioxidant enzymes (SOD, CAT, GST and GPx) activity, thereby increasing the susceptibility of brain and muscle to imminent damage with potent free radicals. But, the oxidative damage was more in the muscle than the brain. In combination, selenium and alpha-tocopherol significantly mitigated the oxidative stress and restored the activities of antioxidant enzymes than individual administration. Based on these findings, this study reports that the selenium and alpha-tocopherol, in combination, effectively mitigated the fluoride-induced oxidative stress in mice models. Further studies are needed to elucidate a specific mechanism.

Acknowledgement

The authors thank the Head, Department of Zoology, UCS, Osmania University, TS, India, for providing laboratory facilities.

Conflicts of interest

The authors declare that they have no conflict of interest for this study.

Funding Support

The authors received financial assistance from UGC-SAP-DSA (F.5-26/2015/DSA-(SAP-II), the programme of Department of Zoology, Osmania University, TS, India.