Effects of Fluoride on Metabolic enzymes of brain and Gastrocnemius muscle of mice: protective role of selenium and alpha-tocopherol
Chandra Shakar Reddy Nallagouni, Pratap Reddy Karnati*
Dept of Zoology, UCS, Osmania University, TS, India.
*Corresponding Author E-mail: pratapkreddyou@gmail.com
ABSTRACT:
The present study aimed to investigate the ameliorative effects of Selenium and Alpha-tocopherol on activities of enzymes involved in metabolic functions, energy production and transfer in the brain and gastrocnemius muscle of mice treated with sodium fluoride. Animals were separated into five groups and treated with Selenium (05 µg/kg BW), Alpha-tocopherol (2mg/kg BW) alone, and in combination for 15 days to evaluate their ameliorative effects against fluoride (20mg/kg BW) toxicity. The obtained results showed a statistically significant (p<0.05) decrease in the body weight, organo-somatic index, protein content, ALAT, AAT, CPK, acid phosphatase and alkaline phosphatase enzymes activities in the fluoride-treated group in comparison with control group indicates oxidative stress induced by fluoride. The effect of fluoride on enzymes of muscle was comparatively higher, which suggests the greater accumulation of fluoride in muscle than brain. These changes were reverted moderately in Selenium and alpha-tocopherol alone treated groups, and significantly (p<0.05) in Selenium + alpha-tocopherol treated group suggests the combinatorial effect of Selenium and alpha-tocopherol in the amelioration of fluoride-induced toxicity. In conclusion, the findings all pointed to the fact that Selenium and alpha-tocopherol showed combinatorial effects in the amelioration of oxidative stress induced by fluoride.
KEYWORDS: Selenium, Alpha-tocopherol, Brain, Muscle, Enzymes.
INTRODUCTION:
Further, fluoride, at a high level, alters the organo-somatic index (OSI), protein content, the activity of antioxidant enzymes and membrane-bound enzymes such as aspartate aminotransferase (AAT), alanine aminotransferase (AlAT)6, creatine phosphokinase (CPK), and the activity of phosphatases includes alkaline (ALP) and acid phosphatases (AP) involved in the active transport of metabolites7 and induces various pathological disorders.
Since decades, several natural substances have been used as protectants owing to their inexpensive, high safety grounds and availability8. Selenium (Se) plays a vital role in various physiological processes including metabolism, immune responses, and fecundity etc and also it exerts various pharmacological effects such as anti-oxidant, antinociceptive, anti-inflammatory etc9,10. Alpha-tocopherol (α-T) plays a crucial role in the regulation of physiological functions and membrane stability in the nervous system11,12. It traps free radicals such as reactive oxygen species and thereby guards cell membrane against the oxidation13,14,15. Combinedly Se and α-T exerts protective effects against various pathological conditions by improving antioxidant and oxidant balance by reacting with free radicals generated in the lipid peroxidation16,17,18. Hence, this study aimed to report the protective effects of Se and α-T individually and combinedly against fluoride-induced metabolic dysfunctions in the brain and gastrocnemius muscle.
MATERIALS AND METHODS:
Chemicals, Animal maintenance and Experimental design:
Fluoride, Selenium and Alpha-tocopherol were procured from Merck Company. The other chemicals used in the study were of all analytical grade. Swiss albino mice (30+2g) were procured from NIFLA, NIN, Hyderabad, TS, India. The animals were kept in animal house under standard conditions (22+2oC, 12 hrs light-dark cycle, standard pellet diet and water). The animals were separated into five groups (G) (6 animals each). G-I: Saline, Group-II: NaF (20mg/kg BW), Group-III: Selenium (5µg/kg BW), Group-IV: α-tocopherol (2 mg/kg BW) Group-V: NaF+Se+α-tocopherol (20mg/kg bw+5 µg/kg bw+2mg/kg bw). The doses were administered daily between 8.30am– 9.30am for fifteen days. All the experiments were carried out as per the institutional ethical committee guidelines (No: 383/01/a/CPCSEA).
Physical parameters:
Bodyweight and tissue somatic index:
The bodyweight of all the animals was noted before treatment and before sacrificing the animals. The weight of the brain and gastrocnemius muscles was noted. From these values the organo-somatic index was calculated19.
Biochemical assays:
The total protein content was assayed according to the Lowry et al., 1951 method20. The creatinine phosphokinase enzyme activity was estimated by using Gerhardt et al., 197921 method. The activities of aspartate and alanine aminotransferases were assayed by the method of Retiman and Frankel, 195722. The activities of acid and alkaline phosphatases were assayed by the method of Taussky and Shorr, 195323.
Statistical analysis:
The resultant data were analysed using one-way ANOVA followed by Turkey’s studentized range test (HSD). The significant level was considered at p=0.05.
RESULTS:
Bodyweight:
The body weight was significantly (p<0.05) decreased (11.17%) in NaF treated group as compared to the control group, whereas it was restored moderately in NaF+Se, NaF+ α-T and notably (p<0.05) in NaF+Se+α-T treated groups with respect of 8.39%, 7.27% and 1.05% suggesting the combinatorial effects of Se and α-T against NaF toxicity (Fig.1).
Organo-Somatic Index:
The organo-somatic index was significantly (p<0.05) decreased in the brain (11.76%) and muscle (31.03%) in NaF treated group as compared to the control group, whereas it was restored moderately in NaF+Se, NaF+α-T and notably (p<0.05) in NaF+Se+α-T treated groups with respect of 10.29%, 11.02% and 4.41% in brain and 26.72%, 30.17% and 5.17% in muscle suggesting the combinatorial effects of Se and α-T against NaF toxicity (Fig.2).
Figure 1: Effect of Se and α-T treatment on body weight of mice subjected to NaF treatment. Height of histogram bar represents mean values with error bars showing values of 6 animals each. *p<0.05 as compared to control group and #p<0.05 as compared to NaF treated group.
(a)
(b)
Figure 2: Effect of Se and α-T treatment on OSI of the brain (a) and muscle (b) in mice subjected to NaF treatment. Height of histogram bar represents mean values with error bars showing values of 6 animals each. *p<0.05 as compared to control group and #p<0.05 as compared to NaF treated group
Protein content:
The protein content was significantly (p<0.05) decreased in the brain (19.49%) and muscle (22.34%) in NaF treated group as compared to the control group, whereas it was restored moderately in NaF+Se, NaF+α-T and notably (p<0.05) in NaF+Se+ α-T treated groups with respect of 10.62%, 15.93% and 4.40% in brain and 16.48%, 18.81% and 3.52% in muscle suggesting the combinatorial effects of Se and α-T against NaF toxicity (Fig.3).
(a) (b)
Figure 3: Effect of α-T and Se treatment on the protein content of the brain (a) and muscle (b) in mice subjected to NaF treatment. Height of histogram bar represents mean values with error bars showing values of 6 animals each. *p<0.05 as compared to control group and #p<0.05 as compared to NaF treated group.
Creatinine phosphokinase:
The CPK activity was significantly (p<0.05) decreased in the brain (28.49%) and muscle (35.65%) in NaF treated group as compared to the control group, whereas it was restored moderately in NaF+Se, NaF+α-T and notably (p<0.05) in NaF+Se+ α-T treated groups with respect of 22.64%, 26.46% and 12.97% in brain and 33.72%, 32.38% and 18.36% in muscle suggesting the combinatorial effects of Se and α-T against NaF toxicity (Fig.4).
Acid phosphatase:
The acid phosphatase was significantly (p<0.05) decreased in the brain (23.72%) and muscle (28.04%) in NaF treated group as compared to the control group, whereas it was restored moderately in NaF+Se, NaF+α-T and notably (p<0.05) in NaF+Se+ α-T treated groups with respect of 22.03%, 22.15% and 1.69% in brain and 21.79%, 23.73% and 1.94% in muscle suggesting the combinatorial effects of Se and α-T against NaF toxicity (Fig.5).
(a) (b)
Figure 4: Effect of Se and α-T treatment on CPK activity of the brain (a) and muscle (b) in mice subjected to NaF treatment. Height of histogram bar represents mean values with error bars showing values of 6 animals each. *p<0.05 as compared to control group and #p<0.05 as compared to NaF treated group.
(a) (b)
Figure 5: Effect of Se and α-T treatment on AP of the brain (a) and muscle (b) in mice subjected to NaF treatment. Height of histogram bar represents mean values with error bars showing values of 6 animals each. *p<0.05 as compared to control group and #p<0.05 as compared to NaF treated group.
Alkaline phosphatase:
The alkaline phosphatase was significantly (p<0.05) decreased in the brain (9.85%) and muscle (10.79%) in NaF treated group as compared to the control group, whereas it was restored moderately in NaF+Se, NaF+α-T and notably (p<0.05) in NaF+Se+α-T treated groups with respect of 7.46%, 8.43% and 1.16% in brain and 8.64%, 9.14% and 5.48% in muscle suggesting the combinatorial effects of Se and α-T against NaF toxicity (Fig.6).
(a)
(b)
Figure 6: Effect of Se and α-T treatment on ALP of the brain (a) and muscle (b) in mice subjected to NaF treatment. Height of histogram bar represents mean values with error bars showing values of 6 animals each. *p<0.05 as compared to control group and #p<0.05 as compared to NaF treated group.
Aspartate aminotransferase:
The aspartate aminotransferase activity was significantly (p<0.05) decreased in the brain (26.98%) and muscle (30.98%) in NaF treated group as compared to the control group, whereas it was restored moderately in NaF+Se, NaF+α-T and notably (p<0.05) in NaF+Se+ α-T treated groups with respect of 23.64%, 24.11% and 1.26% in brain and 29.13%, 29.42% and 2.02% in muscle suggesting the combinatorial effects of Se and α-T against NaF toxicity (Fig.7).
(a)
(b)
Figure 7: Effect of Se and α-T treatment on AAT of the brain (a) and muscle (b) in mice subjected to NaF treatment. Height of histogram bar represents mean values with error bars showing values of 6 animals each. *p<0.05 as compared to control group and #p<0.05 as compared to NaF treated group.
Alanine aminotransferase:
The alanine aminotransferase activity was significantly (p<0.05) decreased in the brain (10.63%) and muscle (16.31%) in NaF treated group as compared to the control group, whereas it was restored moderately in NaF+Se, NaF+ α-T and notably (p<0.05) in NaF+Se+ α-T treated groups with respect of 6.61%, 6.40% and 3.20% in brain and 14.30%, 12.98% and 1.96% in muscle suggesting the combinatorial effects of Se and α-T against NaF toxicity (Fig.8).
(a)
(b)
Figure 8: Effect of Se and α-T treatment on ALAT of the brain (a) and muscle (b) in mice subjected to NaF treatment. Height of histogram bar represents mean values with error bars showing values of 6 animals each. *p<0.05 as compared to control group and #p<0.05 as compared to NaF treated group.
DISCUSSION:
Fluoride potentially affects oxygen metabolism at the cellular level and enhances the reactive oxygen species production, which in turn, involves in pathogenesis of various manifestations including metabolic, neurological and cellular dysfunction6. In the present study, fluoride exposure caused a significant reduction in body weight and organo-somatic index in comparison with control animals (Fig.2) indicates a decrease in organ weight concerning body weight and this may be due to low food intake, weakening of muscle strength and degeneration of tissue proteins or organ structure in fluoridated animals24,6,7. Reduction in the body weight and organo-somatic index may be due to decreased protein synthesis by fluoride25. In the present study, the decreased protein content in the brain and muscle of fluoride group supports the decreased body weight and organo-somatic index by fluoride toxicity. The results are in agreement with a decrease in protein content was the causative factor for the reduction in body weight and organo-somatic index after fluoride exposure24. A decrease in protein content in the present study could be due to the oxidative stress-induced changes in cellular proteins leading to cellular dysfunctions. Cellular dysfunctions alter the activity of membrane-bound enzymes activity and also the enzymes involved in metabolites transport. In the present study, the metabolic enzymes such as CPK, AAT, ALAT, and metabolite transport enzymes such as AP and ALP activity was significantly altered in the fluoridated group in comparison with the control group. These results are in conformity with the previous studies reporting changes in those enzyme activities after fluoride toxicity6,7. All the changes were moderately reversed in Se and α-T alone treated groups and significantly in the combinedly treated group. The protective effects of Se and α-T could be due to their potent antioxidant activities13,26,27.
CONCLUSION:
In conclusion, fluoride exposure significantly altered the body weight, organo-somatic index, protein content and metabolic enzyme (CPK, AAT, ALAT, AP and ALP) activities, and which were significantly restored by Se and α-T combination than individual's effect. Based on these results, this study emphasizes that the selenium and alpha-tocopherol, in combination, significantly ameliorated the fluoride toxic effects in mice models. Further studies are needed to elucidate the exact mechanism.
ACKNOWLEDGEMENT:
The authors thank the Head, Department of Zoology, UCS, Osmania University, TS, India, for providing laboratory facilities.
FUNDING SUPPORT:
The authors received financial assistance from UGC-SAP-DSA (F.5-26/2015/DSA-(SAP-II), a programme of Department of Zoology, Osmania University, TS, India.
CONFLICTS OF INTEREST:
None.
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Received on 23.02.2021 Modified on 23.06.2021
Accepted on 24.09.2021 © RJPT All right reserved
Research J. Pharm. and Tech. 2022; 15(6):2627-2632.
DOI: 10.52711/0974-360X.2022.00439